Profiles of Drug Substances, Excipients and Related Methodology vol 11, Volume 11 (Analytical Profiles of Drug Substances and Excipients)

Analytical Profiles of

Drug Substances Volume 11 Edited by

Klaus Florey The Squibb Institute for Medical Research New Brunswick, New Jersey

Contributing Editors

Gerald S. Brenner Glenn A. Brewer, Jr. Lester Chafetz Nicholas DeAngelis

Lee T. Grady Hans-Georg Leemann Joseph Mollica Milton D. Yudis

Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences

Paris

ACADEMIC PRESS 1982 A Subsidiary of Harcourt Brace Jovanovich,Publishers New York London San Diego San Francisco SPo Paulo Sydney Tokyo Toronto

EDITORIAL BOARD

Norman W. Atwater Rafik Bishara Gerald S . Brenner Glenn A. Brewer, Jr. Lester Chafetz Nicholas DeAngelis John E. Fairbrother Klaus Florey

Salvatore A. Fusari Lee T. Grady Boen T. Kho Hans-Georg Leemann Joseph A. Mollica Bruce C. Rudy Milton D. Yudis

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ISBN 0-12-26081 1-9 PRINTED IN THE UNITED STATES OF AMERICA 82 83 84 85

Y 8 7 6 5 4 3 2 1

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

E . Abignente, University of Naples, Naples, Italy H . Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia A. A . AZ-Badr, King Saud University, Riyadh, Saudi Arabia I . A . Al-Meshal, King Saud University, Riyadh, Saudi Arabia M . A . Al-Yahyu, King Saud University, Riyadh, Saudi Arabia S. L. Ali, Zentrallaboratorium Deutscher Apotheker e. V., Eschborn, Germany N. W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey D. M. Baaske, American Critical Care, Chicago, Illinois R. Bishara, Eli Lilly and Company, Indianapolis, Indiana G. S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania G. A. Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick, New Jersey P. de Caprariis, University of Naples, Naples, Italy J. E . Carter, Ortho Pharmaceuticals, S o m e d e , New Jersey L. Chajetz, Warner-Lambert Research Institute, Morris Plains, New Jersey N. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania W. DeWitte, Ciba-Geigy, Suffern, New York H . A . El-Obeid, King Saud University, Riyadh, Saudi Arabia A. A. Elazzouny, National Research Center, Dokki, Cairo, Egvpt J. Fairbroth, Stiefel Laboratories Ltd., Sligo, Ireland K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey S . A. Fwari, Parke-Davis, Inc., Detroit, Michigan L. T. Grudy, The United States Pharmacopeia, Rockville, Maryland M . M. A. Hassan, King Saud University, Riyadh, Saudi Arabia J. G. Hoogerhde, Schering-Plough Corporation, Bloomfield, New Jersey vii

viii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, A N D REVIEWERS

J. H . Johnson,American Critical Care, Chicago, Illinois H . Kudin, The Squibb Institute for Medical Research, New Brunswick, New Jersey B. T. K b , Ayerst Laboratories, Rouses Point, New York F. Kreuzig, Biochemie GmbH, Kundl, Austria H. G. Leemunn, Sandoz, Basel, Switzerland E. A. Lo@, King Saud University, Riyadh, Saudi Arabia M. E. Mohamed, King Saud University, Riyadh, Saudi Arabia 1. MoZZica, Ciba-Geigy Corporation, Summit, New Jersey F. I. Muhtadi, King Saud University, Riyadh, Saudi Arabia V. D. Reij, Wyeth Laboratories, Philadelphia, Pennsylvania B. C. Rudy, Mary Kay Cosmetics, Dallas,Texas C. M . S h r m , Wyeth Laboratories, Philadelphia, Pennsylvania D. H . Sieh, The Squibb Institute for Medical Research, New Brunswick, New Jersey H. Stober, Ciba-Geigy, Suffern, New York K. D. Thkker, The United States Pharrnacopeia, Rockville, Maryland T. D. Wilson, Sterling Winthrop Research Institute, Rensselaer, New York B. E . Wyka, Schering- Plough Corporation, Bloomfield, New Jersey M. D. Yudis, Schering- Plough Corporation, Bloomfield, New Jersey

PREFACE

It is now well over a decade that I perceived the need to supplement the official compendial standards of drug substances with a comprehensive review of pertinent physical, chemical, and analytical data and methods. Ten years ago the first volume of Analytical Projiles of Drug Substanceswas published under the auspices of the Pharmaceutical Analysis and Control Section of the APhA Academy of Pharmaceutical Sciences. That we were able to publish one volume per year is a tribute to the diligence of the editors to solicit monographs and even more so to the enthusiastic response of our authors, an international group associated with pharmaceutical firms,academic institutions, and compendial authorities. I would like to express my sincere gratitude to them for making this venture possible. I am pleased to report that five years ago a companion series entitled Phurmacological and Biochemical Properties of Drug Substances was initiated by Morton E. Goldberg under the auspices of the section on Pharmacology and Toxicology, APhA Academy of Pharmaceutical Sciences. So far, three volumes have been published. Over the years, we have had queries concerning our publication policy. Our goal is to cover all drug substances of medical value and, therefore, we have welcomed any monographs of interest to an individual contributor. We also have endeavored to solicit profiles of the most useful and used medicines, but many in this category still need to be profiled. Starting with this, the eleventh volume, we shall also supplement previously published profiles with new data as we can find volunteers to write such s u p plements. In this volume, five of the original profiles in Volume 1have been updated. The goal to cover and update all drug substances of medical value with comprehensive monographsis still a distant one. I estimatethat only about a quarter of such compounds have been profiled so far. We would very much like to accelerate

ix

X

PREFACE

the rate of publication and hope that even more authors can be encouraged to write profiles. All those who have found these profiles useful are requested to contribute monographs of their own. We, the editors, stand ready to receive such contributions.

Klaus Florey

AMINOPHYLLINE Kailus D.Thakker and Lee T.Grady

1.

2.

3. 4.

5.

6.

I. 8. 9.

Description 1 . 1 Nomenclature 1.2 Formula, Molecular Weight, and Composition 1.3 Appearance, Color, and Odor Physical Properties 2.1 Spectral 2.2 Other Properties Methods of Preparation Stability-Degradation 4.1 Stability in Solution 4.2 Stability in Solid State Methods of Analysis 5.1 Identification Tests 5.2 Gravimetric Methods 5.3 Titrimetric Methods 5.4 Spectroscopic Methods 5.5 Chromatographic Methods 5.6 Immunoassays Metabolism Biopharmaceutics and Pharmacokinetics Toxicity References

Analytical Profilcs of Drug Substances Volume I I

1

2 2 2 2 3 3 3 9 9 9 9 10 10 11 11 13 13 26 31 31 33 34

Copyright 01982 by The American Pharmaceutical Association ISBN 0-12-260811-9

KAILAS D. THAKKER AND LEE T. GRADY

2

1.

Description 1.1

Nomenclature 1.11

Chemical Name

Aminophylline is chemically known as 1Hpurine-2,6-dione, 3,7-dih dro-l,3-dimethyl-, compound wTth 1,2-ethanediamine (2:l).

P

1.12

Adopted Names

Aminophylline was also known as theophylline ethylenediamine and euphylline. 1 1.13

Trade Names

Aminophylline is known as carena; inophylline; metaphylline; theophyldine; aminocardol; ammophylline; cardiocilina; cardophyllin; phylcardin; tefamin; cardiomin; grifomin; minaphil; peterphylline; stenovasan; the drox; diophylline; genophylline; phyllindon and theolamine.

P

1.2

Formula, Molecular Weight, and Composition:

11

Anhydrous:

C16H24N1004

420.44

Dihydrate:

C16H28N1006

456.44

Theophylline Ethylenediamine 1.3

85-87% 12-15%

Appearance, Color and Odor

Aminophylline is available in the anhydrous form or as the dihydrate. The dihydrate occurs as white or slightly yellowish granules or powd r. It has a faint ammoniacal odor and a bitter taste. f

AMINOPHYLLINE

2.

3

Physical Properties 2.1

Spectral 2.11

Infrared

The infrared spectrum of aminophylline in mineral oil mull, obtained on a B ckman 4250 spectrophotometer, is shown in Figure l.5 It is generally consistent wi h the reported infrared spectrum of theophylline.5,4 The stretches for -NH2 in ethylenediamine and -NH in theophylline appear as a broad band (combined with the mineral oil signal) in the region of 3.0-4.0 P M . Other signals are in the same vicinity as those for theophylline. The fingerprint region beyond 8.5 IJM is distinctive and can be used for identification. 2.12

Ultraviolet

Spectral characteristics of aminophylline solutions in the ultraviolet region were reported by Andrade an Inacio.' Absorption max ma occurred at 243-5 nm = 1701 and at 273-5 [E:im= 5001 in pH 9.5 borate buffer. LE1cm

''

Figure 2 shows the ultraviolet spectrum of aminophylline in water obtained on a Beckman 5260 recording spectrophotometer. 2.13

Nuclear Magnetic Resonance 2.13.1

Proton NMR

An 80 MHz proton magnetic resonance spectrum of aminoph lline in d6-dimethyl sulfoxide, obtained on a Varian FT-80A,' containing tetramethylsilane as an internal reference, is shown in Figure 3 . It is jimilar to the reported (60 MHz) proton NMR of theophylline. The assignments, based on assignments of theophylline protons, are shown in Table I. 2.13.2

Carbon-13 NMR

The 20 MHz proton-noise decoupled spectrum of aminophylline in d6-dimethyl sulfoxide, obtained on a Varian FT-80A is shown in Figure 4.6 The assignments are shown in Table 11. These are based on assignments of dimethyluracil and 1-methylhypoxanthine.7

t P

WAVELE W T H UM

Fig. 1.

IR Spectrum of Aminophylline

0.8

0.7

a.6

y Q!5

U

z

4

m

g

*1

v)

m

a

0. :

0 .i

.1

240

260

280

300

320

WAVELENGTH (nms)

Fig. 2.

W Spectrum of Aminophylline

5

340

36

l

'

l

~

l

'

l

~

~~ l l ' t \ l

l

10

9

a

I

'

l

6

PPm

Fig. 3 .

H'

NMR of Aminophylline

5

r

l 4

~ 3

l

~ 2

l 1

'

l 0

'

l

~

Solvent I

Fig. 4.

I 3 C NMR of Aminophylline

I

8

KAILAS D. THAKKER AND LEE T. GRADY

Table I Proton Assignment -CH2-N-CH3-

Proton Position (see structure) 10,ll 1 3 7

N-C%N-H C-H

8

Chemical Shift (ppm) 2.75 3.23 3.42 5.67 7.71

aIntensity of signal is proportional to the concentration. This proton may be delocalized in the ring. Table I1 Carbon Assignment

Carbon Position (see structure)

N-CH3 N-CH3

Chemical Shift 29.7 27.5 109.6 148.5 142.7 151.4 155.6

c=c c=c

N-C-N -c=o N-C=O-N

-CH2 on ethylenediamine is buried in the solvent signal as shown in Figure 4. 2.2

Other Properties 2.21

Differential Scanning Calorimetry and Melting Point

The thermogram of aminophylline2 shows two endothermic transitions, one at 120" and another at 272°C. The first transition reflects the melting point of aminophylline; the second transition reflects the melting point of theophylline. Theophylline is known to sublime on melting. 2 2.22

Solubility Aminophylline is soluble in water

AMINOPHYLLINE

(1 g/5 ml) .l

9

It is insoluble in dehydrated alcohol and in

ether.8 3.

Methods of Preparation:

Aminophylline was first prepared by Gruter' by dissolving theophylline in aqueous solutions of ethylenediamine in stoichiometric proportions and evaporating in vacuum over sodium hydroxide. Alternate methods include treating anhydrous or h drated crystals of theophylline with ethylenediamine vapor," and treating a 3 M solution of theophylline in a weak organic base (pyridine, quinoline or M aqueous solution of ethylenea-picoline) with a 2 diamine.11 4.

Stabilitv-Degradation 4.1

Stability in Solution

Solutions of aminophylline become turbid on standing due to absorption of carbon dioxide, with subsequent precipitation of theophylline.' S 8 During the preparation of aminophylline injection, excess ethylenediamine is necessary to keep aminophylline from decomposing.12 4.2

Stabilitv in Solid State

Aminophylline crystals, in the presence of moisture, can absorb carbon dioxide fr m air and decompose into theophylline and ethylenediamine.8 This accounts for its characteristic odor. Mixtures containing aminophylline and ephedrine hydrochloride were found to be discolored13 due to an exchange reaction between the two drugs. The ethylenediamine in aminophylline is presumed to liberate ephedrine base which decomposes rapidly. The color change was accelerated by temperature and humidity. 13

.

Numerous reports are found in the literature on the stability of aminophylline in suppository bases,14-21 especially those containing fatty acids. Dissolution of suppositories made with cocoa butter base was markedly lower than those ma e with macrogol base after storage at 22" for up to a yearaP5 Other physical properties of cocoa butter suppositories such as melting point (Tm) and melting time have been known to increase within weeks of storage at 22"

10

KAILAS D. THAKKER AND LEE T. GRADY

.

and 30" as a result of decomposition16 Increase in the in vitro melting point (above 37" in some casf$) was correlated with poor rate of release of theophylline. Decomposition of aminophylline suppositories is presumed to be due to the formation of insoluble amides ethylenediamine and fatty acids of the suppository b base. etwe'' our decomposition products that have been isolated" were identified as mixturf6 of amides of oleic, palmitic, lauric and myristic acids. Further characterization of the decomposition products by IR GLC, and TLC confirmed the presence of alkyl amides. 20"" Stabili rs such as hydroxylamine hydrochloride have been useful. %f

5.

Methods of Analysis

5.1

Identification Tests

Spectral identification tests include IR, W,Mass and N M R spectroscopy. The melting point of theophylline liberated from aminophylline is the basis of one of the compendia1 identification tests. 22 The following color reactions are also useful identification tests; many are used for dosage forms and for mixtures of pharmaceuticals. (i) Ethylenediamine present in aminophylline reacts with sodium rhodizon85e to form a water-soluble, violet-colored precipitate. Ethylenediamine must be released from aminophylline and converted to acetate or hydrochloride. 0

+2

a+

0

Ethylenediamine also foryz a yellow (ii) precipitate with 2,4-dinitrochlorobenzene. Primary aliphatic and aromatic amines interfere with the test.

AMINOPHYLLINE

11

(iii) Aminophylline develops an orange color with ferric chloride and a yellow color with Ehrlich’s reagent. Color de elopment is rapid. This test was designed by Cooper” €or identification of pharmaceuticals in tablets. (iv) Reaction between dimethylglyoxime and oxidized solutions of purines to form a colored product is the basis of the identification test developed by Kido. 26 (V) Reaction of theophylline with potassium chlorate in hydrochloric acid, followed by exposure to ammonia vapors, produces a purple residue. This reaction is the basis of on of the compendia1 identification tests for aminophylline.2 5

(vi) Bratton-Marshall: Aminophylline in acid solution, when treated with diazotized p-aminobenzene-sulfonic acid or p-aminonitrobenzene produces a yellow or a red color, respectively.27 (vii) Addition of 1-2 drops of a 5% solution of sodium nitroprusside to a solutig of aminophylline in acetone produces a violet color. (viii) Aminophylline powder, on mixing and heatin with copper sulfate powder, also produces a violet color.99 5.2

Gravimetric Methods

Earlier methods of analysis of aminophylline were based on extraction of theophylline with organic solvents such as chloroform and 2-propg~;4~ followed by gravimetric determination of the residue. 5.3

Titrimetric Methods 5.31

Alkalimet ric

The weakly acidic nature of theophylline lends itself to titrations with alkali. Titrations with alkali have be59 carried out t o Jgsay aminophylline using a potentiometric or colorimetric end-point (thymolphthalein as indicator) in mixtures of pha rmaceutica1s.

KAILAS D. THAKKER AND LEE T. GRADY

12

5.32

Acidimetric

Ethylenediamine's basic nature was used in many early methods of analysi strong acid were carried out. ethylenediamin by solvent extraction31 or ion-exchange ~ h r o m a t o g r a p h 3was ~ followed by titrations with hydrochloric acid using a colorimetric end-point (methyl orange as indicator). 5.33

Argentometric

When theophylline is treated with solutions of silver nitrate, it forms a silver salt that is insoluble in water and soluble in nitric acid solutions. Basej6gtlthis property, several assay methods have been develop including the compendia1 assay for aminophylline. general, silver theophyllate is precipitated, filtered, washed, re-dissolved in nitric acid and titrated with ammonium thio cy Aminophylline resent in mixtures of pharmaceuticals'8:5p;39 and xanthines" can be analyzed. Some presence of amm nia is necessary when precipitating silver theophyllate." It has been suggested that argentometric titrations give higher values due to adsorption of silver ions by the very voluminous silver theophyllate precipitate. 31

In an unusual application, silver (lloAg) nitrate was used and radi metric titration was carried out to analyze aminophylline. 41 5.34

Complexometric

Bosly developed a titrimetric method for aminophylline by Precipitating Hg-(the~phylline)~with mercuric acetate, itrating excess H g H ions with ammonium thiocyana:zd4' Similarly, the Cu salt of theophylline is also precipitated excess copper titrated with ethylenediaminetetraacetate.4y94 5.35

Non-Aqueous

Non-aqueous titrations of aminophylline using sodium methoxide as the titrant have been carried out with dirnethylf~rmamide~~ or ben~ene-methanol~~ as the solvent for aminophylline. A differential titrimetric method for aminophylline was dexgloped using acetous perchloric acid as the titrant. This method allows

13

AMINOPHY LLINE

determination of ethylenediamine and theophylline content in a single titration, and is useful for aminophylline tablets, injections and suppositories. 5.4

Spectroscopic Methods

Aminophylline solutions obey the Beer-Lambert Law for a zqncentration range of 0.5-1.2 mg%.5 Schack and Waxler developed a potentiometric assay for aminophylline in biological fluids using chloroform/2-propano1 extraction to isolate theophylline, followed by W-absorbance measurement at 275 nm. The method of Schack and Waxler was used most extensively for analysis of aminophylline in early pharmacokinetics research.a Several other spectrophotometric methods have been developed for aminophylline in mixtures of pharmaceuticals, each one using t organic solvent to isolate an extraction aminophylline. ''*kg In one case, graphical correction was applied to the W absorbances of mixtures of aminophylline and phen barbital, measured at two wavelengths of maxima.58 In another application, extraction of serum samples with a salt-solvent pair of ammonium sulfate and ch1oroform:hexane (7:3 v/v) was carried out followed b back extraction of theophyllinea into aqueous borate buffer ( PH 9 . 0 ) an measurement of UV absorbance at 275 nm. 51 PlavsicgZ used charcoal extraction to isolate theophyl ine from other interfering substances. Since theophylline was reversibly adsorbed on charcoal, elution with organic solvent was followed by measurement of W absorbance. Determination of aminophylline in the blood of patients was carried out after gfidation with potassium dichromate in an acidic medium, separation of the oxidation product by steam distillation and measurement of W absorbance at 257 nm. 5.5

Chromatographic Methods 5.51

Thin-Layer Chromatography

In addition to systems developed for theo~ h y l l i n e ,several ~ TLC systems have been developed for "Aminophylline and theophylline are indistinguishable at biological pH's. Therefore assays of theophylline in biological fluids are also included here due to their obvious applicability.

Table I11 Thin-Layer Chromatographic Systems for Aminophylline

No.

Solvent for Drug

Stationary Phase

Developing Solvent

Detection Methods

Application

1.

chloroform

Silica gel F254

ch1oroform:acetic acid (100 :20)

W,Dragendorff ' s reagent

Analysis of suppositories

54

2.

water

aluminum oxide

benzene:ethanol (9:l or 9:1.5)

W, iodine, modified Dragendorff ' s reagent

Mixture of pharmaceuticals

55

Silica gel F254

(i) acetone:chloroform:l-butanol:25% ammonium hydroxide (30:30:40:10) (ii) ch1oroform:ethyl ether (9O:lO) (iii) ch1oroform:ethanol (9O:lO)

ferric chloride, iodine

Analysis of mixtures of xanthines

56

Silica gel H

ch1oroform:acetone: methanol (8:l:l)

lJv

Analysis of suppositories

18

4.

-----a

Ref.

Table 111 (contd.) No.

Solvent for Drug

St at ionary Phase

Developing Solvent

Detect ion Methods

Application

5.

-----a

Silica g e l

methylene chloride: methano1:acetic acid (90:10:3)

uv

Analysis of tablets ampuls, suppositories

F254 anot mentioned.

Ref.

57

KAILAS D. THAKKER AND LEE T. GRADY

16

analysis of aminophylline and its dosage forms. lists the methods developed for aminophylline.

Table 111

Zorka et alS8 separated and identified several pharmaceuticals in a mixture including aminophylline using silica gel G plates and neutral, acidic, and alkaline mobile phases. Detection was done by UV. Riechert5' developed a "micro"-TLC method for analysis of theophylline in biological fluids. Kieselgel 60 F-254 DL-"Fertigplatten" thin-layer plates were used and developed with ch1oroform:methanol (9O:lO) for a mixture of caffeine and theophylline and with ethyl acetate:methanol:25% ammonia (80:ZO:lO) for a mixture of theobromine and theophylline present in saliva, plasma or urine. Sensitivity of detection claimed was 25 ng/lO ~ 1 . None of the dietary xanthines or other commonly coadministered drugs appear to interfere. 5.52

PaDer ElectroDhoresis60

Whatman Paper No. 1 was used with a potential gradient of 20 V/cm. Several xanthines were separated. Among these, theophylline and aminophylline were best chromatographed in Britton-Robinson buffer at pH 10. Detection agent was 1% solution of disodium-2-hydroxy-3,6naphthalenedisulfonate. 5.53

Pressurized Liquid Chromatography

Numerous liquid chromatographic methods for determinations of theophy 1 ne n biological fluids are listed in the literature.4,f1-g' The maj ri of the 'l-" few are methods use reverse-phase chromatog p listed that use i er normal-phase p b z ' or ion-exchange chromatography. In many cases, chromatographic conditions h e e n developed for a specific application,QY,8B~'9 since adaptation of reported methods may introduce some inttgference from co-adm nistered drugs such as acetazo&mide, trisulfapyrimidinebl or cephalosporins.

'',"

li9

Table IV lists some of the recent, most cited methods of analysis of theophylline in biological fluids by liquid chromatography.

Table IV No.

Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

Reverse-phase C-18 (ion-pair)

methano1:sodium acetate (pH 4 . 2 ) containing 10 mM tet rabutylammonTum chloride (10:90)

Extraction of plasma with chloroform:2-propanol

Separation of theophylline from paraxanthine.

2.

Reverse-phase C-18 (ion-pair)

acetonitri1e:water containing 10 mM tet rabutylammonium chloride (2.5:97.5)

Urine:

3.

Reverse-phase C-18

ethano1:water (20:80)

4.

Reverse-phase C-18 (p-Bondapak C-18)

acetonitrile:O.Ol M - sodium acetate (pH 4 . 0 ) (1:9); 2 . 0 ml/min flow

1.

(50:50)

pH adjusted before chromatography Serum: ultrafiltration

Ref. No. 61

Analysis of theo62 phylline in urine and serum; separation from other xanthines and metabolites. Assay of theophylline 6 3 in plasma; separation of theophylline from sulfisoxazole and ampic i11in.

Molecular ultrafiltration to remove plasma proteins

Direct injection.

64

Table IV (contd.)

No.

Stationary phase

5. Reverse-phase C-18

Mobile Phase

Pre-treatment of sample

Comments

acetonitri1e:water ( 6 : 9 4 ) ; 3.0 ml/min flow

De-proteinization with 2.5 volumes of acetonitrile

Micromethod--only 10-1.11sample is required.

Ref. No. 65

6.

Reverse-phase C-18 (p-Bondapak C-18)

acetonitri1e:acetate buffer (pH 4 . 0 ) ( 7 : 9 3 )

De-proteinization with aqueous acetonitriles olution

Microscale method-66 only 3 0 p 1 plasma needed for analysis; direct injection.

7.

Reverse-phase C-18 u-Bondapak C-18

methanol:l% propionic acid (20:80)

Extraction with chloroform evaporation; sample redissolved in methanol

50-1.1 1 sample needed.

8. Reverse-phase C-18 (p-Bondapak C-18)

methano1:tetrahydrofuran:water containing 10 d/liter sodium acetate

Same as Ref. 62

p-Hydroxyethyltheo-

Q)

(40:10:50)

67

68 phylline as internal standard; separation from other xanthines and commonly administered antibiotics.

Table IV (contd.) Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

9.

Reverse-phase

methano1:sodium acetate (15:85)

Deproteinization of serum samples with two volumes of methanol

B-Hydroxyethyltheophylline used as internal standard; no interference from antibiotics or metabolites.

69

10.

Reverse-phase

methano1:amonium phosphate

Extraction with organic solvent before analysis

100 u1 of serum sample can be analyzed; theophylline can be analyzed in presence of anticonvulsants.

70

11.

R verse-phase C-18

acet nitrile : acetate buffer (pH 4.0) (9O:lO)

Extraction with chloroform:2-propanol (95:5)

8-Hydroxyethyltheo71 phylline used as internal standard; theophylline recovery was found to be between 71 and 75%.

No.

Ref. NO

Table IV (contd.)

No.

Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

Ref.

NO

12. Reverse-phase C-18

acetonitrile: acetate buffer (pH 4 . 0 ) (12:88)

Extraction of 0.5-0.2 ml of acidified plasma with dichloromethane

13. Reverse-phase C-18

methanol:0.05 M phosphate buffer (pH 4 . 7 ) (12:88 v/v)

Molecular filtration of Method applicable serum to separate proteins to human serum, urine and saliva samples, also separation and quantitation of theophylline and its metabolites.

73

2 0 ~1 of blood are

74

h)

0

14.

Reverse-phase

Separation of xanthines; sensitivity of detection 0.1 mg/ liter

sufficient for analysis; detection by direct-curren t pulse and differential pulse amperometry.

72

Table IV (contd.) No.

Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

15.

Reverse-phase C-8

methano1:buffer (14:86 v/v)

Extraction with 2-propanol:chloroform (25 :75)

Completely automat75 ed analysis. Developed on Technicon "Fast-LC" 200-p 1 sample required. No interference was observed.

sodium acetate: 0.02 M methanol (2:l)-

Adjustment of pH to 5.5

Theophylline, sulfamethoxazole, ampicillin and caffeine well separated.

76

acetonitrile: phosphate buffer (pH 4.8) (1:l); 3.0 ml/min flow; 50"

Same as Ref. 85

Authors found that the retention time of internal standard varied from run t o run when using the conditions described in Ref. #5; column temp. was therefore maintained at 50°C.

77

16.

Reverse-phase RP-8

17. Reverse-phase C-18

Ref. No.

.

Table IV (contd.)

No.

Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

18.

Reverse-phase C-18 Whatman Partisi1 10-ODS

acetonitrile: 10 mM phosphate buffer (10:90)

Protein denaturation by acetonitrile

No pre-column necessary; no interference observed.

78

19.

Reverse-phase C-18 p-Bondapak

methano1:pH 2.0 Precipitation of proteins by 50% v/v trichloroacehydrochloric acid solution (containing tic acid 0.02 M potassium chloride)

Theophylline analyzed in presence of methyl xanthines and caffeine.

79

20 *

Reverse-phase C-18

acetonitri1e:acetate buffer (8:92)

Theophylline well Plasma samples extracted with chloroform:2-propanol separated from paracetamol and (95:s); solvent removed and xanthines. and samples re-dissolved dissolved in mobile phase

21.

Reverse-phase C-18 5 p

Solvent A: water containing .01 M sodium acetate and 0.005 M tet rabu ty1ammonium hydrogen sulfate.

Ion-pair extraction using tetrabutylammonium sulfate and ethyl acetate: chloroform:2-propanol, (45:45:10 v/v) after adjustment of urine pH to 6.0-6.5

N

N

Simultaneous quantitation of theophylline and its major metabolites.

Ref. No.

80

81

Table IV (contd.)

No.

Stationary phase

21. contd.

g

22.

Reverse phase p-Bondapak C-18

Mobile Phase

Pre-treatment of sample

Comments

Proteins precipitated by perchloric acid, supernatant neutralized and injected

50 111 of serum are

Ref. No.

Solvent B: ( 5 0 : 5 0 ) methano1:solvent A gradient elution with 9% solvent B at start, 46% solvent B at end of run (for program see Ref. 81). methanol:10 mM monobasic sodium phosphate (1:4); 0.8 ml/min flow

sufficient; theophylline is well separated from dietary xanthines, caffeine, theobromine and theophylline metabolites.

82

Table IV (contd.)

*

K 3 .

No.

Stationary phase

Mobile Phase

Pre-treatment of sample

Comments

23.

Normal phase column (silica)

ch1orform:dioxane: formic acid (95.5:4.5:0.01 v/v)

Equal volume of saturated ammonium sulfate is added to plasma, and then extracted with chlorof orm:2-propanol (95 :5 v/v); solvent is evaporated and residue redissolved in mobile phase

100 u 1 of plasma 83 are sufficient; theophylline well separated from the metabolites.

24.

Normal phase Lichrosorb Si-60

(i) chloroform: Extraction from plasma 2-propanol: with chloroform 2glacial acetic acid propanol (95:5) (92:7:1) with 40% hexane (ii) Ethylene chloride: methanolic ammonium formate (98:2)

25.

Cation-exchange 0.66% acetic acid Partis i1 SCx column temp. at 5OoC

0.1-ml sample extracted with ethyl acetate

Ref.

NO

Mass spectrometry used for identification.

84

Applicable to plasma or saliva samples; no interference.

85

Table IV (contd.)

No.

26.

Stationary phase

Mobile Phase

Strongly basic anion exchange resin (35% crosslinkage)

acetate buffer; linear gradient from 0-6.0 M, flow 0.72 a / m i n

Pre-treatment of sample

Comments

Ref.

NO Sample filtered to remove particulate matter

General method for W-absorbing compounds in urine; 1 30 compounds tested.

86

KAILAS D. THAKKER AND LEE T. GRADY

26

5.54

Gas Chromatography

Earlier attempts to analyze blood levels of theophylline resulted in evelopment of various gas chromatographic methods. g5h_f0' Most of these methods require extraction and derivatization before chromatography. Table V lists some of the more recent methods. 5.6

Immunoassays 5.61

Enzyme Immunoassay (EMIT)

The enzyme immunoassay (EMIT) developed by Syva Corporation (Palo Alto, California) is the most widely used method for the assay of the0 lline (in biological fluids) in clinical laboratories.

nSs

In principle, theophylline antibodies are preparedlo4 by injecting a solution of bovine gamma globulin linked to theophylline ( o r a chemically similar derivative) into sheep. Theophylline (or a derivative) is also linked to an enzyme, in this case, glucose-6-phosphate dehydrogenase. When a patient's serum containing free theophylline is mixed with a solution of antibodies and enzyme-labelled theophylline, the free drug and the enzymelabelled drug compete for the binding sites on antibodies. The reduction in enzyme activity when bound to antibodies can be monitored by using the proper substrate; in this case NADH. This assa s rapid, specific, and requires small s easily adaptable to ommercial sample size. kinetic analyzers, and can be modified"' to suit the application. Comparison of the EMIT assay with -_ chromat~~t;;~~#c methods shows good agreement between the two assays. Vinet et all2' developed another enzyme immunoassay. In this method, the sample is extracted with chloroform/2-propano1, and back-extracted into aqueous sodium hydroxide. Inhibition of beef liver phosphatase by theophylline is determined at 25" C, using p-nitrophenyl acetate as the substrate in a pH 9.4 2-amino-2-methyl-lpropanol buffer system.

In another application nephelometric, competitive immunoassay was developed. '21 A precipitate was obtained by combining theophylline-antibody complex with a macromolecule, and scattering of light by the precipitate

Table V No.

Stationary phase

1.

3% OV-17 on Gas Chrom Q

Conditions

Column at 230°C; nitrogen-phosphorus detector

Pre-treatment of sample

Comments

Sample extracted with chloroform:2-propanol ( 5 0 : 5 0 ) , evaporation and redissolution of residue in 0.02 M tetrabutylammonium-h yd ro xid e

20 p 1 of plasma are

2.

F.I.D. Silicone stationary phase, 2% SP 2510 DA

Extraction of sample with salt-solvent pair of ammonium sulfate and methylene chloride: hexane:acetic acid (80:20:0.1)

3.

5% OV-225 on 80/100 Gas Chrom Q

Sample extracted with ethyl acetate, and then treated with pentafluorobenzyl bromide for derivatization

Electron-capture detector; column at 250°C

Ref.

NO

93

sufficient; detection sensitivity 100 pmol/liter; theophylline well separated from other xanthines and co-administered drugs. 94

100 p 1 of serum are

sufficient; detection sensitivity 0.1 ug/ml.

95

Table V (contd.) No.

Stationary phase

Conditions

4.

3% XE-60 on

80/100 Gas Chrom Q

5.

3% OV-17 on

100/120 Gas Chrom Q 6.

3% OV-17 on 100/120 Gas Chrom Q

Pre-treatment of sample

Comments

Ref. No.

Electron-capture detector; injector and column at 220"C, detector at 225°C

Derivatization by pentafluorobenzyl bromide followed by column chromatography prior to injection

Sensitivity of detection 5 ng/ml.

Nitrogen detector; column at 240°C

Extraction with tetrahexyl- 25-pl sample is ammonium hydrogen sulfate sufficient. in aqueous sodium hydroxide

97

Nitrogen-phosphorus detector; column at 240°C

Off-column derivatization No interference as follows: Sample is ex- from drugs or tracted with dichlorometabolites. methane, dried and reacted with -N,N-dimethylacetamide, tetramethylammonium hydroxide and 1iodopentane. It is then transferred into cyclohexane: pentane mixture (95: S), the solvent evaporated, and it is redissolved in methanol.

98

96

Table V No.

Stationary phase

Conditions

7.

3% OV-17 on Gas Chrom Q

Flame-ionization detector; column at 190°C

Extraction of sample with ether:dichloromethane: 2-propanol ( 6 : 4 : 1 ) , reextraction of the organic layer with 1 N sodium hydroxide, acidification with phosphoric acid, re-extraction with organic solvent, evaporation and redissolution of residue in tetrapropylammonium hydroxide

99

8.

3% SP 2250 on 100/120 Supelcoport

Flame-ionization detector; column temperature programmed from 160°C to 240°C at 8" C/min

Sample extracted with dichloromethane, evaporated, and dissolved in toluene, butylating agent is then added.

100

h)

\o

Pre-treatment of sample

Comments

Ref. No.

Table V (contd.) No.

Stationary phase

Conditions

Pre-treatment of sample

Comments

Ref. No.

9.

3% OV-17 on 100/120 Chrom W HP column well conditioned

Column temperature programmed from 180°C to 280°C; detector at 280"C, mass spectral source at 150°C

Extraction with chloroform, butylation with tetramethylammonium hydroxide and -N,N-dimethylacetamide

Mass spectrometry using probabilitybased matching.

101

10.

3% SP2250 on 100/120 Supelcoport type 50-50 methylphenyl silicone

Column temperature programmed at 190°C to 3OO0C at 10"/min

Samples are introduced in a flash heater for ethylation

No interference.

102

AMINOPHYLLINE

31

was used to quantitate theophylline content. 5.62

Radioimmunoassay (RIA)

The principle of radioimmunoassay is similar to EMIT, except that in this case decrease in radioactivity is measured. Ra ioimmunoassays f r th ophylline have been developed using 'H-theophylline. lY2 ,123 8-Carboxytheophylline was used to prepare antibodies. There is no interference from endogenous purines or known metabolites of theophylline at the concentrations studied. 122 6.

Metabolism

UsinglJZ4C] aminophylline injection, Caldwell, Monks and Smith determined that the metabolites of aminophylline are the same as those of theophylline. 125,126 These are (i) 3-methylxanthine (ii) 1,3-dimethyluric acid and (iii) 1-methyluric acid. However, the rate and extent of conversion to 1,3-dimethyluric acid and 3-methylxanthine were higher or aminophylline than for theophylline.126 Therefore, "C recovery in urine (0-24 hours) was higher for aminophylline (87%) than for theophylline ( 7 6 % ) . The formation of 3-methylxanthine follows saturation kinetics; therefore, the presence of circulating methylxan ines from The foods affects the elimination of aminophylline. appearance of the other wo metabolites follows first order kinetics. Jenne et all2' determined that 1-demethylation of theophylline to 3-methylxanthine is the dominant reaction determining theophylline concentration in serum. Presence of ethylenediaminelyyst affect this conversion, but how it does is not known.

'"

7.

Biopharmaceutics and Pharmacokinetics

Pharmacokinetics of aminophylline has been studied extensively. Since aminophylline and theophylline are indistinguishable in biological fluids, pharmacokineticists d o not differentiate between the two. lthough the pharmacokinetics of theophylline was reported' earlier, the advent of newer analytical techniques has since led to an extensive amount of work. Aminophyl line is administered orally 129-133 as a 1329133,134,135 Or as a singlesustained-re1 dose capsulg- ordosage tablet, fo 15',130 intravenously (or by infusion) ntramuscularly, or rectally as suppositories or enema. 1389131 Among different aminophylline dosage

lwe

"'

32

KAILAS D. THAKKER AND LEE T. GRADY

forms, rect 1 positories give the widest variation.lf8,fYf Absorption of aminophylline from tablets or capsules is rapid, plasma levels reaching therapeutic range within 1-1 l/2 hours. Sustained-release preparations of aminophylline are used often to maintain theophylline levels within for about 12 hours in treatment of as theraPey51'15'~f39 thma The rectal route is used often with infants and children.

.

Plasma theophylline levels of 5-15 pg/ml (after administrati of aminophylline) are considered safe and toxic symptoms do O'' Although in most ca therapeutic. not appear at levels above 25 ug/ml,Bz8 the patient-tothat individualization of the patient variation is therapy is necessary. In some case intra-patient variation in doselblood levels is observedfh6 during milk levels continuous long-term therapy. Saliva and bre of theophylline are lower than plasma levels, but1 &75PZ$ correlation between plasma/saliva ratio is obtained.

fglhi@

ft5

.'

The pharmacokinetic behavior of aminophylline once it appears in the blood can be described by the same ode1 that The is used to describe theophylline pharmacokinetics volume of distri u i n at steady-state for all ages is 0.45 literlkg'158-E5P except in neonates where it is slightly larger.152 Elimination is rapid, with a half-life of about 6 hours in normal, healthy, non-smoking adults. Among the factors that affect the ation of theophylline fr sma are age,1f'i'P'9 physi lo and 163-18 diet ,165 co-administer disease stat 1'B-p89 time of day,fb6 and smoking habits.fg7di'8s'In a case when any of these factors is operating, the half-life of elimination varies from 5 to 30 hours. Very recently, Monks et have compared the disposition and elimination of theophylline and aminophylline. Elimination of aminophylline was faster than theophylline in the same subjects. Although qualitatively the disposition (and metabolism) of theophylline and aminophylline were similar, the authors claimed there were small but significant differences in rates of elimination and the extent of dose eliminated within 48 hours.

AMINOPHYLLINE

8.

Toxicitv

Toxicity of aminophylline results when bl d levels exceed the level of 20 pg/ml of theophylline.lY8 The severity of toxicity is directly related to plasma levels of theophylli In mild toxicity, symptoms are nausea and vomiting ,1981171 diarrhea, abdominal pain, nervousness, insomnia, tachycardia and headache. serious tachycardia, grand ma1 seizures, cardiac arrythmias may occur In some cases, death may result from acute toxicity. ij4

33

KAILAS D. THAKKER AND LEE T. GRADY

34

9.

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AMINOPHYLLINE

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40

KAILAS D. THAKKER AND LEE T. GRADY

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L. Hendeles, M. Weinberger, and G. Johnson, Applied Pharmacology, Applied T h e r a p e u t i c s , San F r a n c i s c o , CA (1980) p. 131.

104.

K. E. R u b e n s t e i n , R. S . S c h n e i d e r , and E. F. Ullman, 47, 846 ( 1972) . Biochem. Biophys. R e s . Commun. -

105.

J . B. Gushaw, M. W. Hu, P. Singh, J. G. Miller, and R .

S. S c h n e i d e r , C l i n . Chem., 23, 1144 ( 1977) . 106.

J. R. Koup and B. Brodsky, Am. Rev. R e s p i r . D i s . , 117, 1135 (1 9 7 8 ).

107.

J. Y. Chang and R. J. B a s t i a n i , C l i n i c a l Study No. 41, Summary r e p o r t , S p a C o r p o r a t i o n , P a l o A l t o , CA (1977).

108.

V. Henry, J. Deutsch, and G. Lum, C l i n . Chem.,

514 (1978).

24 ( 3 ) , --

109.

A. C a s t r o , J. I b a n e z , W. V o ig h t, T. Noto, and H. Malkus, C l i n . Chem., -24 ( 6 ) , 944 ( 1978) .

110.

D. N. D i e t z l e r , N. Weidner, V. L. T i e b e r , J. M. McDonald, C. H. Smith, J. H. Ladenson, and M. P. L a c k i e , C l i n . Chim. Acta, -1 0 1 (2 - 3) , 163 ( 1980) .

111.

N. Weidner, J. M. McDonald, V. L. T i e b e r , C. H. Smith, G. Kessler, J. H. Ladenson, and D. N. D i e t z l e r , C l i n . Chim. Acta, -97 ( l ) , 9 (1979).

112.

M. O e l l e r i c h , G. W.

113.

N. U r quh art, W. Godolphin, and D. J . Campbell, C l i n .

S y b r e c h t , and R. Haeckel, J. C l i n . 17 ( 5 ) , 299 (1979). Chem. C l i n . Biochem., --

Chem., -25 ( 5 ) , 785 (1979). 114.

H. M. He ick , A. Mohammad, and C. Golas, C l i n . Biochem., -12 ( 2 ) , 68 (1979).

AMINOPHYLLINE

115.

D. A. L a c h e r , J . A. S i n n , J. S a v o r y , and M. R. Wills, Ann. C l i n . Lab. S c i . , -10 (4), 305 (1980).

24 (3), 520 116. H. S t o n e and B. G i l l i l a n , C l i n . Chem., -(1978). 24 (2), 391 (1978). 117. J. P. Long, C l i n . Chem., -118. F. D. Lasky, J. A 1 R a z i , and A. Kramen, C l i n . Chem., 24 (8), 1381 (1978). -119. R. L. Boeckx, E. M. F r i t h , and F. E. Simons, "her. Drug Monit., _1 (l), 65 (1979). 120. B. V i n e t and L. Z i z i a n , C l i n . Chem., -25 (8), 1370 (1979). 121. T. Nishikawa, H. Kubo, and M. S a i t o , C l i n . Chim. Acta, 91 (l), 59 (1979). -122. A. L. Neese and L. F. Soyka, C l i n . Pharmacol. T h e r a p . , 21 (5), 633 (1977). 123. C. E . Cook, M. E. Twine, M. Myers, E. Amerson, J. A. K e p l e r , and G. F. T a y l o r , Res. Commun. Chem. P a t h . Pharm., - 13, 497 (1976).

124. J. C a l d w e l l , T. J. Monks, and R . L. S m i t h , B r . J . Pharmacol., 63, 369p (1978). 125. H. H. C o r n i s h and A. A . C h r i s t m a n , J. B i o l . Chem., 228, 315 (1957). 126. J. W. J e n n e , T. W. C h i c k , B. A. Miller, and R. D. S t r i c k l a n d , Am. J. Hosp. Pharm., 34, 408 (1977). 127. T. J . Monks, R. L. S m i t h , and J. C a l d w e l l , J. Pharm. Pharmacol., 33, 93 (1981). 128. J. W. J e n n e , H. T. Nagasawa, and R. D. Thompson, C l i n . Pharm. and Therap., -19 (3), 375 (1976). 129. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. J. Hosp. Pharm., 34, 525 (1977). 130. M. L. S l o t f e l d t , C. E. Johnson, G. Grambau, and J. G. Weg, Am. J. Hosp. Pharm., -36 (l), 66 (1979).

41

KAILAS D. THAKKER AND LEE T. GRADY

42

131.

H. Lamont, E. Moermann, M. B o g a r t , M. Van Der S t r a e t e n , and R . Pauwels, Eur. J. C l i n . Pharmacol., 1 5 ( 6 ) , 401 (1979). --

132.

G. E. M a r l i n , M. A. B u t c h e r , J. A. Klumpp, and P. J. Thompson, B r . J. C l i n . Pharmacol., -10 (3), 265 (1980).

133.

P. W. Trembath and S. W. Boobis, C l i n . Pharmacol. Ther., 26 ( 5 ) , 654 (1979). - --

134.

S. McKenzie and E. B a i l l i e , J. I n t . Med. Res., Suppl. 1, 22 (1979).

135.

7 -

M. C. Meyer, A. B. S t r a u g h n , and P. Lieberman, C h e s t ,

78 ( 2 ) , 300 (1980). -136.

L. J. Lesko, A. T. Canada, G. Eastwood, D. Walker, and D. R. Brousseau, J. Pharm. S c i . , -6 8 ( l l ) , 1392 (1979).

137.

M. W. Weinberger, R. A. Matthay, E. J. Ginchansky, C. A. Chidsey, and T. L. P e t t y , J. Am. Med. Assoc., 235, 2110 (1978).

138.

E. B. T r u i t t , J r . , V. A. McKusick, and J. C. K r a n t z , J r . , J. Pharmacol. Exp. Therap., 1 6 0 , 309 (1950).

139.

J. Ahrens, Dtsch. Med. Wochenschr.,

(1977).

102 (13), 482 --

140.

M. W. Weinberger and E. A. Bronsky, J. P e d i a t r . , 421 (1974).

141.

E. Ginchansky and M. Weinberger, J . P e d i a t r . , -91 (4), 655 (1977).

142.

P. R a n g s i t h i e n c h a i and R. W. Newcomb, J. P e d i a t r . , 91 ( 2 ) , 325 (1977). --

143.

F. Nielsen-Kudsk, I. Magnussen, T. S . J e n s e n , and K. Naeser, Acta Pharmacol. T o x i c o l . , -46 ( 3 ) , 205 (1980).

144.

F. E . Simons, K. J. Simons, G. G. S h a p i r o , W. E . P i e r s o n , and C. W. Bierman, J. Med., 9 ( l ) , 81 (1976).

84,

43

AMINOPHYLLINE

145. M. H. J a c o b s , R. M. S e n i o r , and G. Kessler, J. Med. Assoc., 235, 1983 (1976).

Am.

J. 146. P. D. Watson, R. C. S t r u n k , and L. M. T a u s s i g , P e d i a t r . , -91 (2), 321 (1977). J. 147. N. N. Khanna, H. S. Bada, and S. M. Somani, Pediatr., 96, 494 (1980).

148. S. P. G a l a n t , S. A. Gillman, L. H. Cummins, P. P. Kozak, and J. J. O r c u t t , Am. J. D i s . C h i l d . , -131 (9), 970 (1977). 149. R. Koysooko, E . F. E l l i s , and G. Levy, C l i n . Pharmacol. Therap., -15 (5), 454 (1974). 150. P. A. Mitenko and R. I. O g i l v i e , C l i n . Pharmacol. Therap., 14, 509 (1973). 151. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. Rev. R e s p i r . D i s . , 118, 97 (1978). 152. 3. V. Aranda, D. S. S i t a r , W. E. P a r s o n s , P. M. Loughnan, and A. H. N e i m s , N. Engl. J. Med., 295, 413 (1976). 153. G. G i a c o i a , W. J. J u s k o , J. Menke, and J. R. Koup, J. Pediatr., 89, 829 (1976). 154. J. P. Rosen, M. D a n i s h , M. C. Ragni. C. L. S a c c a r , S. -~ J. Y a f f e , and H. I. Lecks, P e d i a t r i c s , 64, 248 (1979). 155. F. E. R. Simons and K. J. Simons, J. C l i n . Pharmacol., 18, 472 (1978). 156. P. M. Loughnan, D. S. S i t a r , R. I. O g i l v i e , A. E i s e n , 2. Fox, and A. H. N e i m s , J. P e d i a t r . , 88, 874 (1976). 58, 157. E. F. E l l i s , R. Koysooko, and G. Levy, P e d i a t r i c s , 542 (1976). 158. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I. O g i l v i e , N. Engl. J. Med., 296, 1495 (1977). 159. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I. O g i l v i e , C l i n . Pharmacol. Therap., 21, 310 (1977).

44

KAILAS D. THAKKER AND LEE T. GRADY

160.

A. Mangione, T. E. I m h o f f , R. V. L e e , L. Y. Shum, and W. J u s k o , C h es t , 73, 616 (1978).

161.

N. Vicuna, J. L. McNay, T. M. Ludden, and H. S c h w e r t n e r , B r . J. C l i n . Pharamcol., 7, 33 (1979).

162.

K. C. Chang, T. D. B e l l , B. A. L a u e r , and H. C h a i , L n c e t , 1, 1132 (1978). -a -

163.

R. A. Landay, M. A. G o n z a l e z , and J. C. T a y l o r , J. A l l e r g y C l i n . Immunol., 62, 27 (1978).

164.

M. Weinberger, D. Hudgel, S. S p e c t o r , and C. C h i d s e y , J. A l l e r g y C l i n . Immunol., 59, 228 (1977).

165.

A. Kappas, K. E. Anderson, A. H. Conney, and A. P. Alvares, C l i n . Pharmacol. Ther., 23, 445 (1978).

166.

L. J. Lesko, D. B r o u s s e a u , A. T. Canada, and G . Eastwood, J. Pharm. S c i . , -69 (3), 358 (1980).

167.

J. J e n n e , M. Nagasawa, R. McMugh, F. McDonald, and E . Wyse, L i f e S c i . , 17, 195 (1975).

168.

J. R. P o w e l l , J. T h i e r c e l i n , S. Vozeh, L. Sansom, and S. Reigelman, Am. Rev. Resp. D i s . , 116, 17 (1977).

169.

W. J. J u s k o , J. J. S c h e n t a g , J. H. C l a r k , M. G a r d e n e r a n d A. M. Yurchak, C l i n . Pharmacol. T h e r a p . ,

406 (1978). 170.

24,

L. H e n d e l e s , L. B i g h l e y , R. H. R i c h a r d s o n , C. D. H e p l e r , a n d J. C a r m i c h a e l , Drug I n t e l l . C l i n . Pharm., 11, 12 (1977).

171.

T. R. Kordash, R. G. van D e l l e n , and J. T. M c C a l l , J. Am. Med. Assoc., 238, 139 (1977).

172.

L. W. Z w i l l i c h , F. D. S u t t o n , J r . , T. A. N e f f , W. M. Cohn, R. A. Matthay, and M. W. W e i n b e r g e r , Ann. I n t e r n . Med., 82, 784 (1975).

173.

M.

174.

C. L. Winek, J. D. B r i c k e r , W. D. Collom, and F. W. Fochtman, F o r e n s i c S c i . I n t . , 15 (3), 233 (1980).

S. Schwartz and D. F. S c o t t , E p i l e p s i a , 15, 501 (1974).

ASCORBIC ACID Ibrahim A . Al-Mesh1 and Mahmoud M . A. Hassan

1.

2.

3.

4. 5. 6. 7. 8. 9.

10.

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight I .4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Specific Rotation 2.4 Dissociation Constant 2.5 Identification 2.6 Spectral Properties Preparation 3.1 Isolation 3.2 Synthesis Biosynthesis of Ascorbic Acid Metabolism Daily Requirement Mode of Action Vitamin Deficiency Methods of Analysis 9.1 Titrimetric Methods 9.2 Spectrophotometric Methods 9.3 Turbidimetric Method 9.4 Chromatographic Methods 9.5 Enzymatic Method 9.6 Polarographic Method 9.7 Chronometric Method References

Analytical Profiles of Drug Substances Volume II

46

46 46 47 47 47 47 47 48

48 49 49 51 61 61 63 63 66 66 66 67 67 67 70 73 73 75 15 75 76

Copyrighl 0 1982 by The Amenan

45

phrrm.ceuUcd Awduion ISBN 0-12-260811-9

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

46

1,

Description 1.1. Nomenclature

1.1.1

Chemical Names

a) b) c) d) 1.1.2

L-Ascorbic a c i d L-Xyloascorbic a c i d 3-0x0-L-gluofuranolactone (enol form). L-3-Ketothreohexuronic a c i d l a c t o n e

Generic Names Vitamin C ; A s c o r b i c a c i d

1.1.3

Trade Names Adenex; A l l e r c o r b ; A n t i s c o r b u t i c Vitamin; Ascorbicap; Ascorbajen; A s c o r i l ; A sc o ri n ; A s c o r t e a l ; A s c o r v i t ; Cantan; C a n t a x i n ; Catav i n C ; Ce b i c u r e ; Cebid; Cebione; Cecon; Cegiol an ; Cell i n ; Cenetone; Cereon; Cergona; Ce s c o r b a t ; Cetamid; Cetan; Cetemican; Ceval i n ; Ce v a t i n e ; Cevex; Cevimin; Cevi-Bid; Ce-Vi-Sol; Cevitamin; C e v i t a n ; Cimin; C e v i t a mic Acid; C e v i t e x ; Ciamin; C i p c a ; C o l a s c o r ; Concemin; C-vimin; Davitamon C ; E r i v i t C ; Hybrin; L a r o s c o r b i n e ; Lemascorb; Megascorb; P l a n a v i t C ; Pr o s c o r b i n C ; Redoxon; Ribena; Sc o r b a c i d ; Scorbu-C; T e s t a s c o r b i c ; V i c e l a t ; V i t a c e ; V i t a c i mi n ; V i t a c i n ; V i t a s c o r b o l ; Vitix.

1.2

Formulae 1.2.1

Empirical ‘gH8’6

1.2.2

Structural

CtI*OtI

I

HR=o

HOCH

HO

OH

41

ASCORBIC ACID

1.2.3

CAS No.

(50-81-7) 1.2.4

(Wiswesser Line Notation TOSV EHJ CQ DQ EYQ IQL (1)

1.2.5

Stereochemistry The nature o f the ring system in ascorbic acid was determined by a study of the methylated derivatives of the acid. By this means complete confirmation was obtained of the accuracy o f the views advanced, concerning the stereochemical configuration of the molecule and the nature of the reactive enolic groups(2). The furanose structure for ascorbic acid shown above (1.2.2) was put forward by Lerbert el a1 ( 2 ) on the basis o f its chemical behaviour as well as its oxidation products.

1.3

Molecular Weight 176.12

1.4

Elemental Composition C, 40.91%; H, 4.58%; 0 , 54.51%.

1.5

Qpearance, Color, Odor and Taste White or slightly yellow crystals or powder. Odorless or almost odorless; pleasant sharp acidic taste(3).

2.

Physical Properties 2.1

Crystal Properties Usually plates, sometimes needles, monoclinic system (4). 2.1.1

X-ray Diffraction The available data (2) of the X-ray, reveals that the total of 12 carbon and oxygen atoms

48

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

all but one can be accomodated in one plane without appreciable valency strain whilst the remaining carbon (C5) lies less than 1A" above the plane as shown in the following model :

2.1.2

Melting Range Ascorbic acid melts at 190-192" with decomposition ( 4 ) .

2.2

So lubi1ity

Ascorbic acid is soluble at 20", in 3.5 parts of water and 25 parts of alcohol (95 per cent); 50 parts of absolute alcohol, 100 ml of glycerol, 20 ml of propylene glycol. Solubility in hot water 40.0% at 40°, 80% at 100". Insoluble in ether, chloroform, benzene and light petroleum (boiling range 40-60"). 2.3

Specific Rotation [a] [a]

['I

I'[

is+ i3+

20.5" to 21.5" (C = 1, water) 48 (C = 1, methanol) (4).

l9 + 24 (water) (2). 5780 l8 + 116 (sodium salt in neutral solution) ( 4 ) 5780 l8 + 130 (N/20 NaoH) ( 2 ) . 5780 l8 5780 + 149 (N/l NaoH)

(2).

l8 + 155 (N/2 NaoH) 5780 l8 + 161 (2N NaoH) 5780

(2) (2)

49

ASCORBIC ACID

2.4

Dissociation Constant Ascorbic acid is a moderately strong organic acid, two ionization constants: pK1 4 . 1 7 and pK 11.57. pH = 3 (5mg/ml), pH = 2 2 (50 mg/ml) (4).

2.5

Identification i)

Solution of ascorbic acid decolorises, 2,6dicholorophenol-indophenol solution (5).

ii)

Solution and ascorbic acid reduces silver nitrate solution immediately in the cold producing a black precipitate (5).

iii)

Dissolve 0.1 g of ascorbic acid in sufficient

w a t e r to produce 100 ml and dilute 1 ml to 100 ml with 0.01M h y d r o c h l o r i c a c i d . The

light absorption of the resulting solution exhibits a maximum only at 244 nm; A (1 per cent, 1 cm) at 244 nm, about 560 (6).

iv)

To 2 ml of a 5 per cent w/v solution add 0.5 g of s o d i u m h y d r o g e n c a r b o n a t e ; carbon

dioxide is evolved ( 6 ) .

v)

To 1 ml of a 5 per cent w/v solution add about 0.2 ml of ZM n i t r i c a c i d and 0.2 ml of 0.1M silver n i t r a t e ; a grey precipitate is

produced (6).

vi)

To 5 ml of a 1 per cent w/v solution add 0.05 ml of a freshly-prepared 5 per cent w/v solution of s o d i u m n i t r o p r u s s i d e and 2 ml of 2M s o d i u m h y d r o x i d e followed by 0.6 to 0.7 ml of h y d r o c h l o r i c a c i d , added dropwise with stirring; the yellow color turns blue (6).

vii)

Specific optical rotation, in a 10 per cent w/v solution, +20.5' to +21.5O (6).

viii)

A

solution (1 in 50) reduces alkaline cupric tartrate TS slowly at room temperature but more readily upon heating ( 7 ) .

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

50

ix)

To 2 ml of a solution (1 in 50) add 4 drops of methylene blue TS, and warm to 40": the

deep blue color becomes appreciably lighter or is completely discharged within 3 minutes (7).

XI

Dissolve 15 mg in 15 ml of a solution of trichloroacetic acid (1 in 201, add about 200 mg of activated charcoal, shake the mixture vigorously for 1 minute, and filter through a small fluted filter, returning the filtrate, if necessary, until clear. To 5 ml of the filtrate add 1 drop of pyrrole, and agitate gently until dissolved, then heat in a bath at 50": a blue color develops ( 7 ) .

xi)

For the examination of vegetable infusions

for the presence of vitamin C, the ascending

- descending paper-chromatographic method of

Block was used on the dinitrosazones. Many combinations of solvent were used, but a mixture of xylene and nitrobenzene (95:5) were the most satisfactory. Of these, the latter solvent furnished compact spots of such definition that treatment with alcoholic potash to reveal them was unnecessary (8)

xii)

Amounts of vitamin C down to 3 pg can be detected as dark areas on the layer exposed to short-wave UV light; brief heating to 120°C renders it fluorescent in radiation of 365 nm ( 9 ) .

xiii)

The customary identification o f free ascorbic acid depends on its strong reducing properties and any of the reactions known from paper chromatography may be utilized. The limit of detection with indophenol reagent (blue) is around 0.1 pg dipyridly-iron (red) and molybdophosphoric acid (blue) are almost as sensitive; after brief heating, derivatives and decomposition products yield the colors also. Amounts of 3-5 pg can be visualized with iodoplatinate reagent (yellow) and with alkaline silver nitrate reagent (9).

51

ASCORBIC ACID

2.6

Spectral Properties 2.6.1

Ultraviolet Spectrum The UV spectrum of ascorbic acid (0.002%) in aqueous, acidic methanolic, ethanolic and alkaline solution was scanned from 200 to 400 nm using Varian Carry 119 Spectrophotometer (Fig. I ) . The UV maxima are as follows: 'max Aqueous solution Acidic solution Methanol Ethano 1

(nm)

263 243 244 245

Other reported data (2) are as foJlows: 'max Aqueous solution Acidic solution (pH3) Ethano 1 Methanol Sodium salt in aqueous solution 2.6.2

(nm>

260-265 245 245 263 265

Infrared Spectrum The IR spectrum of ascorb,icacid as KBr-disc was recorded on a Perkin-Elmer 580B FTspectrometer (Fig. 2 ) . The structural assignments have been correlated with the following band frequencies (Table 1).

Table

-

1: IR Characteristics of Ascorbic Acid,

Frequency Cm-I

Assignment

3510 3405 3306

OH

1755 1670

c=o

1110 1025

c-0-c

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

52

.

.

.

200 210 225 230 2 4 250 260 270 284 2YO

300 310 2 0 3%

3u)

350

-

3m 380 390

Fig. 1. UV Spectrum of Ascorbic Acid. Ascorbic Acid i n Water; --- Ascorbic Acid i n Ethanol; +scorbic Acid i n Ascorbic Acid i n Acid Solution. Methanol;

....

4w mnmlmbr(tlDm

Fig. 2.

am

fWe

mr

1

f4a

IR Spectrum of A s c o r b i c A c i d as KBr d i s c .

c

w

I

a

o

c

o

o

4

m

2

I

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

54

Other characteristic absorption bands are: 3208, 1500, 1390, 1372, 1320, 1275, 1250, 1222, 1200, 1140, 1075, 1068, 1045, 1025, 990, 870, 820, 755, 720, 680. 2.6.3

Nuclear Magnetic Resonance Spectra 2.6.3.1

Proton Spectra The PMR spectra of ascorbic acid in dueterium oxide,in pyridine and in pyridine D5 were recorded on a Varian-T-60-A, 60 MHz spectrometer using sodium-2,2-dimethyl-2-silapentane-5-sulphonate and tetramethylsilane as reference standard respectively (Fig.3, Fig. 4 and Fig. 5). The following structural assignments have been made (Table 2):

Table - 2:

PMR Characteristics o f Ascorbic Acid.

Chemical Shift (ppm) D20

Pyridine

Pyridine D5 Assignment

4.87(d) 4.10(m) 3.77(s) 3.68(d)

5.43(d) 4.68(m) 4.40(s) 4.30(s)

5.36 (d) 4.33 (m) 4.36 (s) 4.23 (d)

'

5-H 6-H 7-CH2

s=singlet, d=doublet, m=multiplet.

400

r

Fig.

3.

PMR Spectrum of A s c o r b i c A c i d in D20.

z

0

I m

0

0

0 m 0

1

;I 56

L I

L

m 4

Fig. 5.

PMR Spectrum of Ascorbic Acid i n Pyridine D5.

58

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

PMR data in D20 and in a mixture of DMSO D6 and CDC13 were also reported (10,11,12). 13

2.6.3.2

C-NMR

The 13C-NMR completely decoupled and off-resonance spectra are shown in Fig. 6 and Fig. 7 respectively. Both were recorded over 5000 Hz range in dimethylsulfoxide on Jeol FX-100, 100 MHz spectrometer. Using 10 mm sample tube and tetramethylsilane as reference standard, at ambient temperature. The carbon chemical shifts are assigned on the basis of the additivity principles and the protoncoupled spectrum (Table 3).

11

I HO

- c 6-

I Table

-

3:

Carbon Chemical Shifts of Ascorbic Acid.

Carbon No.

Chemical Shift ppm

c-1 c-2 c-3 c-4 c-5 C -6

170.31(s) 117.93 ( s ) 152.62 (s) 74.56 (d) 68.42 (d) 61.93(t)

s=singlet; d=doublet; t=triplet 13C-NMR data in Water have been also reported (1 3) ,

Fig. 6.

coupled).

13C-NMR Spectrum of Ascorbic Acid (Completely de-

~~

Fig. 7 .

13C-NMR Spectrum of Ascorbic Acid (off-Resonance)

.

61

ASCORBIC ACID

2.6.4

Mass Spectrum The mass spectrum o f ascorbic acid obtained by electron impact ionization, was recorded on a Ribermag R-10-10 mas spectrometer equipped with direct inlet probe. The spectrum (Fig. 8) shows a molecular ion peak M+ at m/e 176 with a relative intensity o f 5.9%. The most prominent fragments and their relative intensities are shown in Table 4:

Table M/e

-

4: Prominent Mass Fragments of Ascorbic Acid. Fragment

Relative Intensity %

177 176

M + l M +

1.0 5.9

1

116

100.0

85

36.0

71

24.7

70

23.1

HF

H

H o g

a' 61 3.

Preparation 3.1

29.7

HO-

C

PH - 0

I H

I H

'i'

C

Isolation Many methods were reported €or the isolation o f ascor bic acid from plants. However, the most popular is by using freshly prepared solution of 5-6% methaphosphoric acid (14). This solution is a good extractant as well as stabilizing agent f o r a limited period by complexing metal ions and minimizing the rate of oxidation. It has also been claimed that ascorbic acid can be stablized by diluted perchloric acid solution

ASCORBIC ACID

63

o r 2,3-dimercapto-l-propanol (15). An alternative method of extracting ascorbic acid from foods is by forming a slurry of the frozen material with absolute ethanol has been found to be as effective as extraction with metabphosphoric acid (16).Also a mixture of 8% acetic acid and 0.5% oxalic acid was used (17) 3.2

Synthesis L-ascorbic acid is conventionally synthesized (18,19) by hydrogenating D-glucose to D-sorbitol. The latter is made to yields L-sorbitol by oxidation with Acetobacter suboxydan, this followed by introducing carboxyl group at C 1 while the L-sorbose is in the form of its diacetone derivative. The resulting diacetone-2keto-L-gluconic acid is then heated with hydrochloric acid t o give ascorbic acid (Scheme 1 , p . 11). Alternative route from sorbose by oxidation with nitrogen peroxide. Another method for synthesizing L-ascorbic acid was reported ( 2 0 ) , involving a one-step oxidation of 1,20-isopropyhene-a-D-glucofuranose to 1,2-0-isopropylidene-a-D-xylo-hexofuranurono-6,3-Lactone-S-ulose and acid treatment of the later followed by reduction.

4. Biosynthesis of Ascorbic Acid In both plants and animals ascorbic acid is formed from Dglucuronic acid. UDP-glucuronic acid is first converted to D-glucuronic acid lactone via D-glucuronic acid-l-phosphate. This compound is then reduced at carbon atom 1 t o form L-gulonic acid. (Since in the numbering of the carbon atoms of carbohydrates the most highly oxidized carbon atom is given the lowest possible number, the original carbon atom 6 of glucuronic acid becomes carbon atom 1 of gulonic acid). After the conversion of the gulonic acid to the corresponding y-lactone, the hydroxyl group at carbon atom 2 is oxidized to a keto group. The 2-keto-Lgulonic acid lactone formed is subsequently converted t o L-ascrobic acid by enolization (21). Direct conversion of D-glucuronic acid to L-gulonic acid by isomerization at carbon atom 5 has not yet been conclusively established (scheme 2,~.12).

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

64

CH20H I HO - C I HO - C -

CH20H

I

H

IIO - C

H

I

I

H?

I

cat.

H -C-O€i

HO

-c - B

-

HO-

C-

B-

C-

HO -

I

CH20H I

H

co

OH

I C - I-;

Acetobactv suboxydans

I

I CHO D-glucose

I

HO-

H

C--B

I

H-

C-

I HO - c I

OH

H

Ct120H

CH20€i

G-sorbitol

L-sorbose COOH

I

co HO acetone H+

(1)*<Mn04

+

I

9:CMe2

I

ki

-

C

HO

-

C

~

( 2 ) H 2 0 (H+)

H

I

- cI

CH OII 2

2-keto-L-gulonic acid

(CH20H C -OH

II enolize

11

C

I

I

lactoniz

H - C-OH C-H I CH20H

bo

H

I

HO-

-E

I

2,3,4,6-diacetone sorbose

HO-

- OH

HO-C-H

OH

I

I

CH20H

L-ascorbic acid (Vitamin C) Scheme 1: Synthesis of ascorbic acid.

O€i

65

ASCORBIC ACID

6

-

0 II ..

Q"DpCOOH

COOH

Ho@o-p

HO

OH

OH

UDP-D-Glycuronic acid

C

,@"20H

D-Glucuronic acid1-P

CH20H

OH

D-Glucuronic acid lactone

CH20H

L-Gulonic acid

L-Gulonic acid lactone

4

I CH20H

2-Keto-L-gulonic acid lactone.

HO-C-H

I

CH20H

L-Ascorbic acid.

Scheme 2: The biosynthesis of L-ascorbic acid.

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

66

5.

Metabolism Ascorbic acid is readily absorbed and metabolised. However, after oral administration of large quantities, only small amounts are excreted in the urine while there is a steady rise in the level of ascorbic acid in the plasma. If the oral ingestion is continued for a sufficient period, the plasma concentration rises to a maximum, after which a rapid urinary excretion of a large part of the ingested ascorbic acid occurs (22). Ascorbic acid (ASA) and dehydroascorbic acid (DAsA) are metabolized by humans, and the levels of AsA in blood were maximum at the 4th hr after AsA administration but at 2nd hr after DAsA administration. The amount of AsA and DAsA excreted in urine in 6 hr was respectively. 60 and 70% of the amount excreted in 24 hr after administration, About 30 and 40% of administered dose were excreted by men and women respectively in 24 hr. Of the vitamin excreted after administration of either AsA or DAsA, about 90 and 10% were in the form of AsA and DAsA, respectively (23). The amino acids phenylalanine and tyrosine are not metabolized completely in vitamin C-deficient individuals, Under these conditions they are metabolized only partly and are excreted in the urine as homogentisic, p-hydroxyphenylpyruvic and p-hydroxyphenyllactic acids. It appears that vitamin C plays the role of a coenzyme in the metabolism of tyrosine through its deaminated product, because scorbutic liver slices cannot metabolize this amino acid in the absence of this vitamin. Vitamin C in adequate amounts delays the oxidation of epinephrine by the body (24 1.

6.

Daily Requirement! Ascorbic acid is needed in daily quantities of about 70 mg to sustain full stamina, and is an essential nutrient for human beings. In the case of insufficiency the symptoms of scurvy appears (21).

7.

Mode o f Action We have as yet no complete understanding o f the mode of action of ascorbic acid. It is, however, known that this substance is involved in certain hydroxylation reactions which are catalysed by mixed function oxygenases in the reduction o f folic acid to tetrahydrofolic acid as well as well in the regulation of the redox equilibrium between Fez+ and Fe3+ and Cu" and C U ~ +(21).

61

ASCORBIC ACID

8. Vitamin Defficiency

Patients placed on a diet deficient in vitamin C exhibited the following: 1) 10 days, plasma fell to a low level; 2) 30 days plasma level was zero; 3) 13 weeks, first clinical evidence of scurvy; 4) 132 days, hyperkeratotic papules developed; 5) 141 days, wounds failed to heal and 6) 162 days, perifollicular hemorrhages of scurvy developed; ascorbic acid value of white cell platelets fell to zero. Loss of weight occurred, accompanied by lowered blood pressure (24). 9. Methods of Analysis 9.1 Titrimetric Methods

British Pharmacopoeia 1973 The B.P. 1975 has described the assay as follows: Weigh and powder 20 tablets. Dissolve a quantity of the powder equivalent to 0.15 g of ascorbic acid as completely as possible in a mixture of 30 ml of water and 20 ml of d i l u t e sulphuric acid and titrate with 0.lN anunonium c e r i c sulphate, using f e r r o i n sulphate solution as indicator. Each ml of 0 . 1 1 m o niwn c e ri c sulphate is equivalent to 0.008806 g of C6H806. British Pharmacopoeia 1980 The B.P. 1980 has described the assay as follows : Dissolve 0.2 g in a mixture o f 80 ml of freshly boiled and cooled water and 10 ml of M sulfuric acid. Titrate with 0 . 0 5 M iodine VS using 1 ml of starch solution as indicator until a persistent blue color is obtained. Each ml of 0.05M iodine VS is equivalent to 0.00881 g of C H 0 6 8 6' United States Pharmacopoeia 1980 a) Ascorbic Acid: Dissolve about 400 mg of ascorbic acid, accurately weighed, in a

68

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

mixture of 100 ml of carbon dioxide-free water and 25 ml of 2N s u l f u r i c acid. Titrate the solution at once with 0.1 N iodine VS, adding 3 ml o f starch TS as the end-point is approached. Each ml of 0.1 N iodine is equivalent to 8.806 mg of C6H806. b)

Ascorbic acid injections: Transfer to a 100-ml volumetric flask an accurately measured volume of ascorbic acid injection, equivalent to about 50 mg of ascorbic acid and previously diluted with water if necessary. Add 20 ml of metaphosphoric-acetic acids TS, dilute with water to volume, and mix. Accurately measure a volume of the dilution, equivalent to about 2 mg of ascorbic acid, into a 50-ml conical flask, add 5 ml of metaphosphoric-acetic acids TS, and titrate with standard d i c h l o r o p h e n o l - i n d o p h e n o l solution until a rose-pink color persists f o r at least 5 seconds. Correct for the volume of the dichlorophenol-indophenol solution consumed by a mixture of 5.5 ml of metaphosphoricacetic acids TS and 15 ml of water. From the ascorbic acid equivalent o f the standard d i c h l o r o p h e n o l - i n d o p h e n o l solution calculate the ascorbic acid content in each ml o f the injection. Various other titrimetric methods of assay of vitamin C in vegetable tissues, whether as ascorbic acid or as total vitamin C (ascorbic plus dehydroascorbic acids), were studied and compared. For the extraction of the vitamin, an aqueous mixture of 8 per cent, acetic and 0.5 per cent oxalic acids was used instead of metaphosphoric acid. Hot and cold extractions gave practically the same results, except with hard, dry tissues, which required hot extraction. To effect removal of colloidal matter, which interferes with filtration and clarification of the extract, it is considered advisable to add 25 to 50 per cent o f ethanol. In the presence of ethanol causes an error o f at least 2 to 12 per cent. To establish (visually) the titration end-point in the indophenol method, it is recommended that a standard having the same composition as the sample under titration

69

ASCORBIC ACID

b u t p r e v i o u s l y o x i d i s e d w i t h i o d i n e and mercuric a c e t a t e be employed. T h i s g e n e r a l l y e n a b l e s 93 t o 100 p e r c e n t of v i t a m i n C e x p e r i m e n t a l l y added t o be determined. I n t h e method of d e t e r m i n a t i o n with methlene b l u e , t h e r a t i o o f methylene b l u e used t o v i t a m i n C c o n t e n t d e c r e a s e s as t h e l a t t e r v a l u e i n c r e a s e s . T h i s d e c r e a s e of t h e r a t i o i s small; t o o b t a i n s a t i s f a c t o r y r e s u l t s , t h e a l i q u o t t i t r a t e d should n o t c o n t a i n >0.04 mg o f v i t a m i n C . V i s u a l d e t e r m i n a t i o n o f t h e end-point w i t h t h e a i d o f a s t a n d a r d i s a g a i n recommended. The i o d i m e t r i c method, with potassium i o d a t e was a l s o s t u d i e d . The c o n c l u s i o n s drawn a r e : 1) t h a t t h e indophenol method i s s u f f i c i e n t l y a c c u r a t e , i s t h e most simple method and h a s t h e w i d e s t s p h e r e of a p p l i c a t i o n ; 2) t h a t t h e methylene b l u e method g i v e s r e s u l t s 3 t o 10 p e r c e n t lower t h a n t h e indophenol method and 3) t h a t t h e i o d i m e t r i c method g i v e s r ? s u l t s 20 t o 40 p e r cmt higher (17). Reduction i s e f f e c t e d by adding M sodium s u l p h i d e s o l u t i o n a c i d i f i e d with HC1, and removing t h e e x c e s s o f s u l p h i d e w i t h M e t h a n o l i c mercuric c h l o r i d e s o l u t i o n . Reduct i o n i s complete i n 10 t o 15 min. C l e a r f i l t r a t e s a r e r e a d i l y o b t a i n a b l e €or t i t r a t i o n w i t h dichlorophenol-indophenol ( 2 5 ) . Simple methods o f d e t e r m i n i n g a s c o r b i c a c i d , t h i a m i n e and n i c o t i n i c a c i d a r e applied t o mixtures containing t h e s e vitamins with various pharmaceutical p r e p a r a t i o n s . Ascorbic a c i d can b e determined i o d i m e t r i c a l l y i n t h e p r e s e n c e o f calcium l a c t a t e , p h y t i n g l u c o s e calcium g l y c e r o p h o s p h a t e , c a f f e i n e sodium b e n z o a t e , n i c o t i n i c a c i d , amidopyrine o r t h i a m i n e . For m i x t u r e c o n t a i n i n g a s c o r b i c a c i d and nicotinic acid, the ascorbic acid i s determined i o d i m e t r i c a l l y and t h e t o t a l a c i d i s

70

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

then titrated against 0.1 N solution of hydrochloric acid with phenol red as indicator. Thiamine i s determined in the presence of ascorbic acid by a modification of the U.S.S.R. Pharmacopoeia VIII argentimetric method ( 2 6 ) . The platinum - tungsten bimetallic electrode system is applicable to the quantitative iodimetric determination of ascorbic acid according to the B.P. 1953. The equivalence point is given by the very sharp break in the titration curve. The error, = f 0.0002 on samples of 0.05 to 0.15 g is much less than for the usual volumetric procedure (27). Other Sitrimetric method for the determination of ascorbic acid is by involving two stage oxidation by potasium iodate ( 2 8 ) . 9.2

Spectrophotometric Method 9.2.1

Colorimetric An assay method described for ascorbic acid involves the reaction with diazotised 4methoxy-2-nitroaniline in acid medium, and subsequent development of a blue color in alkaline solution. This color, with a maximum absorbancy at 570 nm, i s compared with standards in suitable photo-electric colorimeter. It can be carried out directly, e.g., in the presence of dehydro-ascorbic acid and all other vitamins. Its sensitivity permits the determination of quantities down to 0.5 mg with a low limit of 10 pg per ml, when a 50ml sample aliquot is used (29a).

9.2.2

Ultraviolet In aqueous solution ascorbic acid is characterised by a single very intense band with its head at 260-265.nm. The molecular extinction coefficient is approximately 7000 for solutions containing about 2 mg/100 cc. In stronger solutions (ca.50 mg per 100 c.c.) wide deviations from Beer's law are encoun-

71

ASCORBIC ACID

tered. But for concentrations ranging between 0.5 and 2.5 mg per 100 C.C. Beer's law holds with sufficient exactitude to permit of the use of spectrophotometric measurements for quantitative estimations of concentrations. The intensity of the band diminishes rapidly, falling to half value in a few hours (decomposition of ascorbic acid by oxidation (2). Other color reactions have been proposed as a basis for measuring ascorbic acid: reaction with diazotised p-aminobenzoic acid to produce a pink color and the interaction of ascorbic acid with dimethoxydiquinone to form a reddish -violet color which is measured spectrophotometrically at 510 nm (29b). Ascorbic acid, cystine, thioglycollic acid, a-mercaptopropionic acid and sodium mercaptobutane sulphonate are determined by the fluorescence produce by the reduction of sodium 1 : Z naphthaquinone-4-sulphonate (Folin's reagent) in U.V. The use of p-chloromercuribenzoic acid in the determination of ascorbic acid with 2,6-dichlorophenol-indophenol below pH3-5 is recommended because of the extent of spontaneous fading of indophenol that occurs. p Chloromercuribenzoic acid does not affect the reaction between indophenol and ascorbic acid, but it inhibits almost entirely the decolorisation of indophenol by various interfering substances (31). 9.2.3

Spectrofluorimetric The reaction of dehydroascorbic acid with ophenylenediamine to give a fluorescent quinoxaline was used as the basis of an assay to determine y quantities of ascorbic and dehydroascorbic acids, The development of the fluorescent derivative of the vitamin is prevented by forming a boric acid-dehydroascorbic acid complex prior to addition of the diamine solution. This provides a means of differentiating between the fluorescence from the vitamin and that from possible interfering substances. When applied to pharmaceutical preparations, beverages and special dietary foods,

72

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN

the method shows a high degree of specificity.

No interfering substances were found (32).

A rapid colorimetric determination of ascorbic acid (10 to 200 mg per 100 g) with the sodium salt of 2,6-dichlorophenol-indophenol was studied. An amyl acetate extract of 2,6dichlorophenol-indophenol has max. absorption at 525 nm the extinction of which remains unchanged for 4 hrs. The sample is extracted with 2 per cent metaphosphoric acid and diluted to produce a 0 . 4 to 1.0 mg per cent solution of ascorbic acid. 2,6-dichlorophenolindophenol solution (5 ml in water) is added to the sample solution which is rapidly extracted with amyl acetate for the extinction to be measured. Another 5-ml portion of the 2,6-dichlorophenol-indophenol solution is added to metaphosphoric acid and similarly treated. The amount of ascorbic acid is determined from the difference in the extinction of the two extracts, which is propor-. tional to the concentration of ascorbic acid up to 4 mg per 100 ml ( 3 3 ) .

Other method depends on the reduction of ferric chloride by ascorbic acid and the colorimetric determination of the ferric chloride by means of reaction with aa-dipyridly; the reaction is carried out in the presence of phosphoric acid to eliminate interference by reduction. A solution (1 ml) containing N 0.02 mg of ascorbic acid is treted with 0.3 ml of 85 per cent. Phosphoric acid to give a pH of 1 to 2, 5 ml of 0.5 per cent aqueous aa-dipyridly solution and 1 ml of 1 per cent ferric chloride. The method has been applied to orange juice, honey and urine; it is sensitive to 5 lJg of ascorbic acid per ml ( 3 4 ) . A direct method for the determination of vitamin C without removal of the reagent, 2,4dinitrophenylhydrazine, is applied to blood and urine. The ascorbic acid content i s calculated from a chart (35).

Ascorbic acid can be semi-quantitatively estimated with the comparison method after color reaction with molybdophosphoric acid.

73

ASCORBIC ACID

The red zone of dehydroascorbic acid-DNP is scraped off, eluted with 85% sulphuric acid, filtered or, better, centrifuged, and the light absorption of the solution measured at 520 to 525 nm against water as blank. The analysis result is worked out with the help of a standard solution which is treated identically ( 9 ) . 9.3

Turbidimetric Method This method (36) is used €or the determination o f ascorbic acid in foods, by the reaction of selenious acid with ascorbic acid and stannous ions at low pH and room temperature.

9.4

Chromatographic Methods 9.4.1

Paper Chromatography Methods used €or the separation of ascorbic acid by paper chromatography are shown in Table 5 ( p . 21).

9.4.2

Gas-Liquid Chromatography Gerstl and Ranf€t (42) extracted food with metaphosphoric acid, separted the ascorbic acid on a column of cellulose and formed the trimethysilyl ether derivative by reaction with N.0-bis-(trimethylsily) acetamide before chromatographing on a column of Gas Q containing 3% SE-30.

9.4.3

High Pressure Liquid Chromatography Packla and Kissinger ( 4 3 ) , used a strong anion resin and elution with pH 4.75 buffer solution, has been applied to the determination of ascorbic acid in milk products, baby foods, fruit juice concentrates, whole fruits and fortified cereals. The procedure was not directly suitable for measuring dehydroascorbic acid. However, sood et a1 (44) used HPLC in

TABLE - -- -~

~

~~~

Chromatogram

-

5

~

~

Solvent System

Rf Value

Paper Chro- 1) n-butanol saturated matography. with water + oxalic 2) Phenol saturated with water + oxalic acid. Paper (Schleicher and Schull No. 602).

P

Methods used for separating ascorbic acid by paper chromatography

1) 50% Methanol.

0.7-0.8

2) Wat er-n-butano1-

0.6-0.7

glacial acetic acid (50:40:14:5)

Paper Chro- butanol-acetic acidmatography. water (4:1 :5) Paper Chro- n-butanol-acetic acidmatography. water (4:l:S)

0.36

Reagent 2,6-dichlorophenolindophenol with subsequent colorimetry.

Application Reference Plant and animal tissues.

37

1) 1% silver-nitrate Lemon juice 38 solution in 10% white,red and Aqueous ammonia. black-currents. 2 ) 0.005N iodine solu- tomatoes and tion in 0,04% starch green pappers. so 1ut ion. 3) 0.04% aqueous solution of 2,6-dichlorophenol-indophenol. 4) U.V. ammonium-molybdate solution. Alkaline tetrazolium salts.

39

Method f o r de- 40 tection of lfpg quantity.

ASCORBIC ACID

75

the reverse phase ion-pairing mode to determine ascorbic acid in food with tridecyl ammonium formate as counter-ion. 9.5 Enzymatic Method A new enzymatic method based on the oxidation of the ascorbic acid to dehydroascorbic acid with ascorbic oxidase permit assaying for ascorbic acid and dehydroascorbic acids in vegetable extracts (45). 9.6

Polarographic Method Ascorbic acid can be analysed in a variety of mixtures and multivitamins preparations by cathode ray reverse sweep polarography (46).

9.7 Chronometric Method The reduction oxidation couple between ascorbic acid oxidation and reduction of the semiquinoid form pphenylenediaimine is the basis of a new chronometric assay of vitamin C. Ascorbic acid interrupts formation of the colored product by coupling its oxidation to reduction of the semiquirioid proceeds to dye form (47).

76

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

10. References

1. G.C.G. Grasselli and N.M. Ritchey "Atlas of Spectral Data and Physical Constants for Organic Compounds" 2nd Ed. , Vol. 2, CRC Press Inc., Cleveland, Ohio, 1975. 2. R.W. Herbert, E.L. Hirst, E.G.U. Percival, R.J.W. Reynolds and Smith, J. Chem. SOC., 1270 (1933). 3. J.E. Hoover "Remington's Pharmaceutical Sciences", 15th Ed., Mack Publishing Company, U.S.A., (1975). 4. M. Windholz "The Merck Index" 9th Ed. Merck and Co., Inc., Rahaway, U.S.A. (1976). 5. "The British Pharmacopeia", Her Majesty's Stationary Office, Cambridge, (1973). 6. "The British Pharmacopeia", Her Majesty's Stationary Office, Cambridge, (1980). 7. "The United States Pharmacopeia", XX, Mack Publishing Co., Easton, Pa (1980). 8. R.C.R. Barreto, Rev. Quim. Ind., Rio de Janeiro, 24, 13, (1955). Through, Anal. Abstract , 3, 567 (1956). 9. E. Stahl , "Thin Layer ChromatograpFy", 2nd Ed. , SpringerVerlag., p. 304, (1966). 10. C.J. Pouchert and J.R. Campbell "The Aldrich Library of NMR and Spectra" Vol XI (1974). 11. "High Resolution NMR Spectra Catalog", Varian Analytical Instrument Division, Vol. 2, The National Press, U.S.A. (1963) . 12. D.T. Sawyer and J.R. Brannan, Anal. Chem., 38 (2), 192, (1966). 13. S . Berger, Tetrahedron, 33, 1587 (1977). 14. R.D. King "Developments in Food Analysis Technique-1" Applied Science Publishers, 18 (1978). 15. C.B. Bourgeois, P.R. Mainuy, R. George and A.M. Czornomaz, Analusis, 2, 556 (1973). Through Reference 14. 16. V.G. Randall, E.L., Pippen, A.L. Potter and R.M. McCready, J. Fd. Sci., 40, 894. Through Reference 14. 17. G, Enachescu,%al. Inst. Cerc. Agron. Romn., 22, 463, (1955) . Through Anal. Abstract , 3, 3474 (1956): 18. T.A. Geissman, "Principles of Organic Chemistry", 3rd. Ed., W.H. Freeman and Company, p. 497, (1968). 17, 311, 19. T. Reichstein and A. Grussner, tlelv. Chim. Acta., (1934). 20. J. Bakke and 0. Theander Chemical Comm. , 175 (1971). 21. M. Luckner "Secondary Metabolism in Plants and Animals", Science Paperbacks, p. 74, (1977). 22. E.G.C. Clarke "Isolation and Identification of Drugs", The Pharmaceutical Press, London, (1971).

.

ASCORBIC ACID

77

23. T. Masaru, W. Sanae, M. Katsuko, T. Sachiko, F. Akji (Biochem. Lab. Kagawa Nutr. Coll., Tokyo, Japan). Vilamins 45(3), 136, 1974, fJapan). Through 24. C.D. Wilson, 0. Gisvold and R . F . Doerge "Textbook o f Organic Medicinal and Pharmaceutical Chemistry", 7th Ed., J.B. Lippincott Co., Philadelphia, (1977). 25. E . Piyanowski, Prezem. Rony Spozywezy, 11,410 (1954). 26. G.A. Vaisman and S.G. Rozhntskaya Aptechnoc Delo. (3), 16. Through: Anal. Abstract, 2, 563 (1955). 27. S.R. Mohanty, K.R.K. Rao and L.V. Kannan, Anal. Chim. Acta, 14 (6) 587 (1956). Through: Anal. Abstract, 2, 347, (1956); 28. G.S. Deshmukh and M.G. Bapat, Z. Anal. Chem. 145(4), 256 (1955). 29. (a) M. Schmall, C.W. Pifer and E.G. Wollish, Anal. Chem. 25(10), 1486 (1953). Through Anal. Abstract, 1,370, (1954). (b) M.H. Hashmi, M.A. Shahid, M.A. Akhtar and N.A. Chugtani, Mikrochim Acta, 5, 901 (1973). 30. H.. Freytag, Z. anal. Chem., 139(4), 263, 1953. Through. Anal. Abstract, 1, 371, (1954). 31. J.A. Owen and B.Iggo, Biochem. J., 62(4), 675 (1956). 32. C. Mike, J. Deutch and C.E. Weeks, J. Assoc. Office Agr. Chemists, 48(6), 1248 (1965). Through Chem. Abstract, 64, 72840 (1966). 33* H. Imai and T. Fugitani J. Chem. SOC., Japan, Pure Chem. Sect., s(11), 1212 (1955). Through: Anal. Abstract 2, 2567 (1956). 34. M.X. Sullivan and H.G.N. Clarke, J. Ass. Off. Agric. Chem., 38(2), 514 (1955). Through: Anal. Abstract, 2, 258 (1956). 35. J.H. Roc and C.A. Kuether, J. Biol. Chem. 147, 399 (1943). Through: Chem. Abstract, 37, 31185 (1943). 36. W.J. Ralls, J. Agric. Food Chem. 23(3), 609, (1975). Through: Chem. Abstract, 83, 41610 F (1975). 37. Yu-Tuan Cheng., F.A. Isherwood and L.W. Mapson, Biochem. J., 55(5), 821 (1953). Through: Anal. Abstract, I, 372, (1954). 38. V. Sanda Ceskosl. Farmac., 2(3), 79 (1954). Through: Anal. Abstract, 2, 2563, (1955). 39. W. Hermann, R. Strohecker and F. Matt., Z. Lebensmitt. Untersuch. U. Forsch., 97, 263, (1953). Through: Anal. Abstract, 1, 369, 1954. 40. 2. Padre, E. Smid and V. Sicho, Naturwissenschaften, 42(8) , 210, (1955). Through: Anal. Abstract, 2, 853 (1956). 41. J.E. Schlack, J. Ass. Office, Analyt. Chemist., 57, 1346, (1974). Through: Reference 14. 42. R. Gerstl and K. Ranfft, Z. Lebensmitt-elunters. U. Forsch., 154, 12, (1974). Through Reference 14.

78

IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN

43. L . A . Packla and P.T. Kissinger, Anal. Chem., 48, 3 6 4 , ( 1 9 7 6 ) . Through Reference 1 4 . 4 4 . S.P. Sood, L.E. Sartori, D.P. Wittmer and W.G. Haney, Anal., Chin., 4 8 , 796, ( 1 9 7 6 ) . Through Reference 1 4 . 4 5 . A. Marchesini, F . , Montauori, D. Muffats, D. Maestri, J. Food Sci., 3 9 ( 3 ) , 5 6 8 , ( 1 9 7 4 ) . Through Chem. Abstract, 8 2 , 2717 F ( 1 9 7 5 ) . 4 6 . K S . Owen, F.W. Franklin, J. Food Technol., E ( 3 ) , 2 6 3 , ( 1 9 7 5 ) . Through Chem. Abstract, 83, 1 3 0 1 0 2 J ( 1 9 7 5 ) . 4 7 . B. Roe, J . H . Bruemmer, Proc. Fla. State Hortic. SOC., 87, 210, ( 1 9 7 5 ) . Through Chem. Abstract, 83, 130100 G (1975).

CAPTOPRIL Harold Kadin

1.

2. 3. 4.

5.

6.

7

8.

9. 10. 11.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor History Synthesis Physical Properties 4.1 Spectral Properties 4.2 Solid State Properties 4.3 Solution Data Stability 5.1 Solid State Stability 5.2 Solution Stability Analytical Tests and Methods 6.1 Elemental Analysis 6.2 Spectrophotometric Methods 6.3 Chromatographic Methods 6.4 Titrimetric Methods Analysis in Biologic Fluids and Tissues and in Animal Rations 7.1 Thin Layer Radiochromatography (TLRC) 7.2 Gas Chromatography-Mass Spectroscopy (GC-MS) 7.3 Gas Chromatography-Flame Photometric Detection (GF-FPD) 7.4 High Performance Liquid Chromatography with UV Detection (HPLC-UVD) 7.5 Spectrofluorometry 7.6 Radioimmunoassay (RIA) 7.7 Semiautomated Ellman Colorimetry 7.8 High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ECD) 7.9 High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FD) Drug Metabolism-Pharmacokinetics 8.1 Blood Level Studies 8.2 Urinary Excretion Studies 8.3 Miscellaneous Distribution Studies Acknowledgments References Review Coverage Dates

Analylical Profilca of Drug Substances Volume 11

79

80 80 80 80 84 84 84 97 106 107 107 108 112 112 112 113 120 120 120 122 123 124 124 124 125 126 128 128 128 129 134 131 131 137

Copynghi 0 1982 by The American Pharmaceutical Association ISBN 0-12-260811-9

80

1.

HAROLD KADIN

Description

1.1 Name, Formula, Molecular Weight Captopril, Capoten@, or Lopirins is l-(3-mercapto-2-D-methyl-l-oxopropyl)-L-proline (S,S) with Chem. Abstr. Registry Number 62571-86-2.

The asterisks indicate the two S,S optically active centers. 1.2

Appearance, Color, Odor

Captopril is a white to off-white crystalline powder with a slight mercaptan odor. 2.

History

The captopril story began in 1971 with a report (1) on the isolation and synthesis of teprotide, an antihypertensive nonapeptide from the venom of a Brazilian Pit Viper. This venom peptide was hypotensive through inhibition of an exopeptidase, known as the angiotensin converting enzyme (ACE). The latter performs dipeptide scission at the carboxyl end of the decapeptide, angiotensin I to yield angiotensin 11, the most powerful natural vasoconstrictor known. ACE further potentiates hypertension through scission-inactivation of the nonapeptide vasodilator, bradykinin. Clinical investigations with both teprotide (2) and captopril ( 3 ) have implicated ACE as the key enzyme in human hypertensive diseases. The first ACE inhibitor shown to be clinically efficacious against hypertension was the synthetic venom peptide, teprotide ( 2 ) .

CAPTOPRlL

81

However teprotide was expensive and only effective parenterally. A simpler, orally effective ACE inhibitor was desired. Analogs of the snake venom hypotensive peptides were quantitatively rated for their in vitro inhibition of ACE (4) and for their effect o n e contractile properties of guinea pig ileum ( 5 ) . The ratings aided construction of a hypothetical active site for ACE (Figure 1). This model was based primarily on the similarity of the enzymatic properties of ACE to those of carboxypeptidase A (CASE A) even though the latter yields amino acids rather than the dipeptides of the former. Further, the active site of CASE A, like ACE contains zinc but unlike that of ACE had been structurally characterized, in the crystalline state, by x-ray diffraction (6). Thus the elaboration of the hypothetical ACE active site and the possibility of a simple ACE inhibitor were stimulated by a 1973 report (7) on, D-2-benzylsuccinic acid, a simple inhibitor, of CASE A. Specific points of inhibitor attachment within the real CASE A site were extrapolated to that within the hypothetical ACE site ( 8 ) . On the basis of these extrapolations, synthesis of simple ACE inhibitors were initiated in 1974. As each of a great number of candidates was quantitatively rated in the aforementioned -in vitro screens (previously developed for the venom peptide studies), the hypothetical site was verified and refined. In the refined model schematically represented in Figure 1, three dimensional amino acid configurations within the catalytic site provided suitably spaced subsites or multiple points of attachment to substrate or inhibitor. The semi-circular clefts in the figure represent hydrophobic subsites which may interact with the lipophilic side chains of inhibitor or substrate. The latter are also putatively bound to the ACE site via the X-H group at their nonscissile terminal peptide bond. As indicated, one of the first simple ACE inhibitors was patterned after all of the venom peptide inhibitors in sharing a terminal proline (succinyl-L-proline). The choice of proline was also influenced by the superior biological stability exhibited by teprotide with

82

HAROLD KADIN

ENZYME

INHIBITOR

RELATIVE IN VlTRO INHIBITION

CAABOXYPEPTIDASE A

D-2-EENZYLSUCCINIC ACID

-7 FH2 ?-

0 = CCH,-CH-C -C = 0

ANGIOTENSIN-CONVERTING ENZYME

SUCC1NYL.L-PROLINE

1

D-2-METHYLSUCCINYL.L-PAOLI"

15

CAPTOPRIL

14000

F i g . 1. Key steps i n t h e d e s i g n o f a s p e c i f i c i n h i b i t o r of t h e a n g i o t e n s i n c o n v e r t i n g enzyme.

CAPTOPRIL

its two terminal prolines (9). In an emulation of D-2-benzylsuccinic acid, but with appropriate lengthening of the chain, the nitrogen and carbonyl of the penultimate "venom peptide" amide linkage were substituted, respectively, with a peptidase-inhibitory methylene and a highly anionic, zinc binding carboxyl. As indicated in Figure 2 the analogy was then extended to the better snake venom inhibitors bearing a penultimate alanine in addition to the terminal proline (D-2-methylsuccinyl-L-proline) . The largest increase in inhibitory activity (about 14 thousand fold over succinyl-L-proline) was obtained when the zinc binding carboxyl was replaced with a thiol which has considerably greater affinity for zinc. The snake venom peptide, teprotide, does not have comparable affinity for zinc, however its binding is enhanced by additional interactions of its amino acid side chains with "clefts" on the enzyme beyond the active peptidase site (10). In short, the orally active antihypertensive drug, captopril, was announced to the scientific community in 1 9 7 7 (11). It was made generally available for treatment of hypertensive diseases in 1 9 8 1 (12,841. Clinical investigations (13) suggest it is also highly efficacious in the treatment of congestive heart failure. Its uniquely designed highly specific affinity for the active site of ACE has resulted in a high ratio of clinical success with a relatively low index of side effects. It has been effective where conventional antihypertensive therapies fail or have an untoward number of side effects.

83

84

3.

HAROLD KADIN

Synthesis

A process (14) is presented (Figure 2) in a chemical reaction sequence which follows this brief description. Methacrylic acid (I) is condensed with thiolacetic acid (11) to give racemic 2-methyl-3acetylthiopropionic acid (111). L-proline is then acylated with the acid chloride (IV) of the thioester (111). The resulting proline thioester (VI) is resolved from its R,S - isomer by aqueous crystallization. Saponification of compound VI with sodium hydroxide affords the sodium salt of captopril which after acidification yields captopril (VII)

.

4.

Physical Properties 4.1

Spectral Properties 4.11

Infrared Spectra

The infrared spectrum of captopril in chloroform is presented in Figure 3 and as a KBr pellet in Figure 4. The infrared spectrum in the latter indicates the presence of the following frequencies and their structural assignments (15). cm-1 1750 1725 1640 2560

Assignment C = 0 (COOH group) C = 0 (amide) S - H

DiffereQfes in the fingerprint regions (1350-900 cm ) of the mineral oil mull infrared spectra of batches 3 and 4 (Figure 5 and 6, respectively) indicate that the low melting batch 3 and the high melting batch 4 are polymorphs (see Section 4.21)

.

85

CAPTOPRIL

Figure 2 Chemical Reaction Schematic Diagram

+

I

CH2 = CCOOH MW = 86.01 Methacrylic

I1

I11

+

AcSH MW 76.11 Thiolacetic

AcSCH2CHCOOH

MW = 162.20

-+

Acetylthioisobutyric

IV

AcSCH2CHC

\

c1

COOH

COOH

MW = 180.65

MW = 259.32 Proline Thioester

VI

VI I

MW = 115.13 Acid Chloride" L-Proline

"'?

H3C 0

AcSCH2CHCN

d)

COOH

COOH MW = 217.28

Proline Thioester

Captopril

0

II

Ac = CH3C(a) Reflux (b) SOCl , DMF, Distillation Ci C12 Wash, HC1, (c) H 0 + NaHCO ci!ystallizaiG.on (d) H20 + NaOH, HC1, CH2C12 extraction

41.

86

Ll

0 0

c

rl

c

u

.ti

k ld

a fd

c

a 4J

ul ro

a,

5 0

X

Ll

.ti

d

a 0

4J

a Id u 0

w

5 Ll

LI

a

a,

i, m

a 0

a c

m k

Q)

c

4J

4J

c

ro

H

a,

4J rl r(

k

a, PI

m

c

M -4

ld

ak ld

c

a &

a, [o

7

0

X Li

a 0

ld

PI

4J

u u-l 0

k

4J 0

a,

a

a,

a

5

2 k

2 rl

wI

PI

a,

k

2

c

v)

4

a

a, k

id k

44

..

G

lJ

H

k

(I)

&

c

H

WAVELENGTH (MICRONS)

3500

F i g u r e 5. Instrument:

2500

2Ooo

lsbo

1600

1400

FREQUENCY (CM-’)

1200

I n f r a r e d S p e c t r u m of C a p t o p r i l , B a t c h 3 , Mineral O i l M u l l Perkin-Elmer,

Model 6 2 1

200

WAVELENGTH (MKRONS)

F R E w € w (CM-')

Figure 6. Instrument:

Infrared Spectrum of Captopril, Batch 4, Mineral Oil Mull Perkin-Elmer, Model 621

HAROLD KADIN

90

4.12

Nuclear Magnetic Resonance Spectra 1

The 270 MHZ H-NMR spectrum of captopril in CDCl is shown in Figure 7. The spectrum was obtained from the University of Chicago courtesy of Professor Josef Fried. Spectral assignments are shown in Table 1. The 13C-NMR of captopril in CD OD is shown in Figure 8. The spectrum was obtained on a Varian Associates XL-100 NMR spectrometer equipped with a Nicolet TT-100 data system. Major peaks are assigned in Table 2 . Minor peaks arise from the presence of cis-trans isomerism at the amide bond (16).

Table 1 Proton-NMR Data for Captopril Proton COOH CL-CH B-CH2 Y-CH,L 6 -CH2 9 -CH-C -STHA -CH3 L SH

'

Chemical Shift ( 6 )'PPM

from TMS (ext.

9.81 4.60 2.03 2.07 3.63

(m) (m)i 2.25 (m) (m) (m)

2.44 2.82 1.17 1.53

(d,q) J=6,9 (m) (d) J=6 (ad) J=9,8

(s)

multiplicities: d=doublet; q=quartet; m=multiplet. J=proton-proton coupling constants in Hertz.

0 F

I

CJ

k

X 5:

a, ZJ

x

..k m

c

4J

$ (II

k 4J

c

H

f *

4

92

i

0 0 4 I I 4

X

c a

L! rd

-d

3

.. c, d

$ k

m

4J

c

H

93

CAPTOPRIL

4

Table 2 Carbon-13 NMR Data f o r C a p t o p r i l i n CD30D. Carbon #

Chemical S h i f t ( 6 )

1 ppm from TMS

175.69 59.84 30.03 25.49 48.24 174.91 43.1 17.07

28.1 R e f e r e n c e d from c e n t e r peak o f t h e C D 3 0 D m u l t i p l e t a t 4 9 . 0 ppm 4.13

Ultraviolet Spectra

S p e c t r a of c a p t o p r i l i n a q u e o u s m e t h a n o l , ( F i g . 9 ) w a t e r , 0.1M sodium h y d r o x i d e and 0 . 1 M h y d r o c h l o r i c a c i d ( F i g . 101, a r e p r e s e n t e d (17). These s p e c t r a d e p i c t a n end a b s o r p t i o n s l o p e w i t h o u t peak o r s h o u l d e r . The s l o p e s p e c t r u m i n 0 . 1 M sodium h y d r o x i d e was s h i f t e d c o n s i d e r a b l y towards h i g h e r wavelengths. S i n c e weak s u l f h y d r y l a b s o r p t i o n i s r e p o r t e d ( 1 8 ) i n t h e 220-230 nm r e g i o n , t h i s a b s o r p t i o n s h i f t may b e d u e t o i o n i z a t i o n o f t h e s u l f h y d r y l f u n c t i o n by t h e a l k a l i . This s h i f t towards higher wavelengths w i t h i n c r e a s e i n pH h a s been u s e d by O n d e t t i ( 1 9 ) t o d e t e r m i n e t h e pK o f t h e s u l f h y d r y l i n captopril (Section 8.32).

HAROLD KADIN

. I

;

. !. !. .. . . ./ i ! I

. . . . . .

0 1

. . . .

.

.

.

.

.

.0,

'

.a,*

,

,

,

4

,

-

Fig. 9. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n 10%aqueous methanol s o l u t i o n . Instrument: Cary 15.

Fig. 10. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n H20, 0.1E H C 1 and 0.1N NaOH Instrument: Cary 15

CAPTOPRIL

95

The spectra suggest that there is a maximum at about 200 nm, attributable to the thiol function. However, precise determination of the peak absorbance was difficult because the extremely large blank absorbances prevented maintenance of a stable balance at this wavelength. Consequently the peak maximum is uncertain in Figure 9. However the peak absorbance is valid in Figure 15 in which the solvent was an HPLC mobile phase (System 4 - Table 8 - Section 6.32). 4.14

Mass Spectrum

The mass spectral pattern indicated in Figure 12 was obtained (20) on an AEI MS-9 Mass Spectrometer. The fragmentation, responsible for the spectrum in Figure 12+ is depicted schematically in Figure 11. The M of m/z 217 and the other fragments are consistent with a sulfur-containing compound of the composition C 9 H1 5NO 3 S (Section 1.1). Fiaure 11 Mass Spectral Fragmentation Schematic Captopril

170

103

m/z 199

+

m/z 202

+ CH3

F

M+ m/z 2 1 7 , 1 m / z 1{3

I

J

L m / z 171

H

172+m/z

m/z 70

r m / z 140

+

b m / z 126

+ CH2SH

H20

+ C02

SH

+ S C H 2 4 m/z 127 + C02

HAROLD KADlN

96

6640

SQ14225 BFlTCH ttNNQ23NB

90.> F U

II)

Z W

t-

Z

H

W

>

80-

78.-

7 50

H

t-

a _I

w

L l

Figure 12. Instrument:

Mass Spectrum of Captopril AEI MS-902 Spectrometer Equipped with Frequency Modulated Tape Recorder, Spectrum Processed o n Digital Equipment Corporation PDP-11 Computer

CAPTOPRIL

4.2

97

Solid State Properties 4.21

Polymorphism

An unstable, low (86OC) melting and a stable, high (106OC) melting form of captopril have been observed. These forms exhibited different unit cells (Section 4.26) on single crystal X-ray examination, differences in their powder X-ray (Section 4.27), and differences in the solid state infrared spectra (Figures 5 and 6). Agreement of their optical rotations, infrared in solution and bioassays established them as polymorphs.

4.22 Differential Thermal Analysis (D.T.A. 1 DTA of the high melting polymorph ( 2 2 ) yielded a sharp, well-defined endotherm at 106OC whereas the low melting polymorph produced a sharp endotherm at 86OC. When the low melting polymorph was allowed to resolidify and the DTA repeated, the endotherm at 86OC had disappeared and an endotherm at 106OC appeared. The latter suggests that the high melting form is the stable polymorph. A DuPont 900 Thermoanalyzer programmed for a temperature rise of 15' per min was utilized for these thermograms. 4.23

Melting Range

The U.S.P. (Class 1) melting range for the high melting polymorph was 105.2 - 105.9' ( 2 1 ) . This agrees well with its D.T.A. endotherm of 106OC. The low melting polymorph has a melting range of 87-88', in agreement with its DTA endotherm of 86O. 4.24

Differential Scanning Colorimetry (D.S.C.)

Use of DSC as a purity index for captopril is supported by titrimetric assays (17) of the carboxyl function (alkalimetry) and of the sulfhydryl function (iodimetry). For instance for batch 4 these yielded 99.6% for the carboxyl and 99.2% for the sulfhydryl in very good agreement with the DSC of 99.7% mole 8 ( 2 2 ) .

HAROLD KADIN

98

4.25

Hygroscopidity

Under ordinary conditions captopril is not hygroscopic. Equilibrium moisture studies (23) indicate no moisture pickup by captopril up to 50% relative humidity at room temperature. Above 50% R.H. it shows a tendency to cake after one to two days. Captopril did not exhibit any visual physical changes and remained dry from 0 to 67% R.H. on exposure for 14 days. Samples exposed to 81% R.H. for 14 days appeared moist (24). 4.26

Single Crystal X-ray Diffraction

Single crystal X-ray analyses have been completed (25) for both the low (melting range 86-87OC) and high (melting range 105-106O) melting polymorphs. Both forms are orthorhombic with the following crystal data: High melting polymorph a = 6.834(2), b = 0 8.821(2), c = 17.982(4)A; V = 1084A, " 3 space group P2 2 2 wiFh four molecules3per unit cell; calRefined to R = 0.04 culakeh density = 1.33 gcmfor the 745 observed single crystal intensities.

A.

.

Low melting polymorph a = 9.496(3), b -= 0 03 space group 12.304(3), c = 19.282(5)A; V = 2253A; P2 2121 witF eight molecules egr unit cell; Refined to R = calculated density = 1.28 gcm 0.06 for the 1093 observed single crystal intensities. B.

.

The structure in both has the S,S absolute configuration with a 2 ( T r a n s ) conformation about the N-C(0) amide bond (the O-C-N-C(2) dihedral angles vary from -4 to +6O). The molecular conformation differ in detail, most notably in the conformation about the (S)C-C(C0) bond. Atomic coordinates relative to orthogonal axes for the high melting form are:

99

CAPTOPRIL

S

N1

c2 c3 c4 c5 C6 06A 06B c7 07 C8 c9 c10

-7.010 -6.499 -5.799 -6.274 -7.067 -7.511 -6.188 -7.165 -5.394 -6.215 -5.3 08 -7.041 -6.163 -8.052

1.595 -1.729 -2.755 -2.492 -1.228 -1.024 -4.132 -4.364 -5.106 -1.576 -2.253 -0.624 0.238 -1.432

-5.766 -1.938 -1.152 0.282 0.252 -1.144 -1.626 -2.286 -1.204 -3.227 -3.754 -4.050 -4.947 -4.821

It was predicted that salt formation with resultant dissociation to a carboxylate anion would influence Capoten to crystallize in its less common E ( c i s ) conformation. This prediction was tested (25) by performing single crystal analysis on the dicyclohexylamine salt of captopril. The analysis indicated that the salt was indeed in the E conformation, i.e. the carbonyl groups of the amide and carboxyl functions are cis to each other. 4.27

Powder X-ray Diffraction

The stable, higher melting polymorph and the lower melting, metastable polymorph are shown in the powder X-ray patterns Figures 13 and 14 respectively (26). The values given in the patterns are also listed in Tables 3 and 4 for the high and low melters respectively. The tables also show the relative intensities (based on peak areas) of the various peaks. A powder X-ray pattern taken on the low melting polymorph after it was heated to about 95OC showed conversion to the stable form.

The X-ray pattern was taken with copper Ka, nickel filtered X-radiation.

100

a a u 0

w

k a, 4J

0

E

a,

P I

L

I !

'

i

I

i

101

0

PI

Table 3 Powder X-Ray Diffraction Data f o r Figure 13 (High Melting Polymorph) 2-13 (DEG.)

9.99 11.35 14.24 16.45 17.21 17.98 19.25 19.85 20.78 22.23 24.52 25.03 25.97 26.56 28.26 29.79 30.81 31.66 33.45 34.28

D(ANGSTROMS)

8.85 7.80 6.22 5.39 5.15 4.93 4.61 4.47 4.27 4.00 3.63 3.56 3.43 3.36 3.16 3.00 2.90 2.83 2.68 2.61

PEAK

22.7 37.9 15.2 21.0 57.1 47.5 34.4 109.1 37.7 49.8 24.1 15.3 62.6 13.7 68.7 10.6 8.4 9.1 8.3

17.9

REL. PEAK

0.208 0.347 0.139 0.192 0.523 0.435 0.315 1.000 0.346 0.456 0.221 0.140 0.574 0.126 0.630 0.097 0.077 0.083 0.076 0.164

AREA

88.1 138.4 122.3 77.5 169.6 166.8 146.3 324.5 130.1 161.7 86.4 60.2 326.6 68.2 243.1 102.0 42.5 43.9 47.9 98.2

REL. AREA

0.270 0.424 0.374 0.237 0.519 0.511 0.448 0.994 0.398 0.495 0.265 0.184 1.000 0.209 0.744 0.312 0.130 0.134 0.147 0.301

Table 3 (Continued) ~ - ~ ( D E G . ) D(ANGSTROMS)

PEAK

36.17 36.34 37.53 38.63

14.8 13.3 9.3 15.3

2.48 2.47 2.40 2.33

REL.

PEAK

0.136 0.122 0.085 0.140

AREA

62.3 37.5 64.9 73.3

REL.

AREA

0.191 0.115 0.199 0.224

Sorted Data (Highest Peak First) z-O(DEG.) 19.85 28.26 25.97 17.21 22.23 17.98 11.35 20.78 19.25 24.52 9.99

D(ANGSTR0MS) 4.47 3.16 3.43 5.15 4.00 4.93 7.80 4.27 4.61 3.63 8.85

PEAK

109.1 68.7 62.6 57.1 49.8 47.5 37.9 37.7 34.4 24.1 22.7

REL.

PEAK

1.000 0.630 0.574 0.523 0.456 0.435 0.347 0.346 0.315 0.221 0.208

AREA

324.5 243.1 326.6 169.6 161.7 166.8 138.4 130.1 146.3 86.4 88.1

REL.

AREA

0.994 0.744 1.000 0.519 0.495 0.511 0.424 0.398 0.448 0.265 0.270

Table 4 Powder X-Ray Diffraction Data f o r Figure 1 4 (Low Melting Polymorph) 2 - 8 (DEG.) 8.71 9.31 11.77 12.71 13.13 15.00 17.21 17.89 18.23 18.40 19.51 20.10 20.61 20.87 21.38 22.14 23.25 23.50 24.10 24.86 25.20

D (ANGSTROMS) 10.15

9.50 7.52 6.96 6.74 5.91 5.15 4.96 4.87 4.82 4.55 4.42 4.31 4.26 4.16 4.02 3.83 3.79 3.69 3.58 3.53

PEAK

24.5 13.7 14.3 14.0 16.6 31.8 14.7 14.0 39.5 34.8 16.9 13.1 30.6 13.1 13.0 62.5 13.9 12.1 16.8 12.5 17.4

REL. PEAK 0.392 0.219 0.229 0.224 0.266 0.509 0.235 0.224 0.632 0.557 0.270 0.210 0.490 0.210 0.208 1.000 0.222 0.194 0.269 0.200 0.278

AREA

68.7 26.3 89.1 43.0 69.4 142.4 43.2 36.8 101.0 77.2 91.2 57.3 112.0 34.8 33.8 265.5 69.1 34.4 113.4 43.4 83.3

REL.

AREA

0.259 0.099 0.336 0.162 0.261 0.536 0.163 0.138 0.380 0.291 0.343 0.216 0.422 0.131 0.127 1.000 0.260 0.130 0.427 0.163 0.314

Table 4 (Continued) ~-WDEG.)

25.88 27.33 28.35 28.94 30.05 34.47

D(ANGSTROMS)

PEAK

3.44 3.26 3.15 3.09 2.97 2.60

17.8 14.6 12.8 14.4 14.0 16.6

REL. PEAK

0.285 0.234 0.205 0.230 0.224 0.266

AREA

105.0 90.5 33.3 104.1 72.8 99.3

FEL.

AREA

0.395 0.341 0.125 0.392 0.274 0.374

Sorted data (Highest Peak First) 2-B(DEG.) 22.14 18.23 18.40 15.00 20.61 8.71 25.88 25.20 19.51 24.10 34.47 13.13 17.21

D(ANGSTR0MS) 4.02 4.87 4.82 5.91 4.31 10.15 3.44 3.53 4.55 3.69 2.60 6.74 5.15

PEAK

62.5 39.5 34.8 31.8 30.6 24.5 17.8 17.4 16.9 16.8 16.6 16.6 14.7

REL.

PEAK

1.000 0.632 0.557 0.509 0.490 0.392 0.285 0.278 0.270 0.269 0.266 0.266 0.235

AREA

265.5 101.0 77.2 142.4 112.0 68.7 105.0 83.3 91.2 113.4 99.3 69.4 43.2

REL.

AREA

1.000 0.380 0.291 0.536 0.422 0.259 0.395 0.314 0.343 0.427 0.374 0.261 0.163

HAROLD KADlN

106

4.3

Solution Data 4.31

Solubility

Captopril at 25OC is freely soluble (1 to 10 parts solvent to 1 part solute) in water, methanol, ethanol (SD3A), isopropanol, chloroform, or methylene chloride. However, it is only soluble (10-30 parts solvent to 1 part solute) in ethyl acetate (27). The solubility of captopril in water, at 250CI is 160 mg/ml (28). A solubility-temperature profile of captopril in water obeyed a linear equation up to 4OoC (28). Beyond this temperature captopril showed extraordinarily high water solubility. Solubility in sesame and corn oils was less than 1 mg/ml at 25OC, whereas the solubility in the synthetic oil triacetin (glyceryl triacetate), at 25OC, was greater than 20 mg/ml (29). 4.32

pKa

-

The pK of the carboxyl of captopril (pK ) is reported (23) to be 3.7. Whereas a carboxyl break was readily observed with alkali potentiometry, the sulfhydryl break could not be detected (17). Therefore, the pK of the sulfhydryl in captopril (pK ) was not estfmated by classical potentiometgy. It was, however, estimated at 9.8 (pK2) by Ondetti (19) and Weiss (30) using sulfhydryl U.V. shifts to higher wavelengths with increase in pH (Section 4.13). The method utilized was adapted from Benesch and Benesch (31). 4.33

Metal Complex Formation

Captopril was modelled (11) as a selective and competitive inhibitor of the angiotensin converting enzyme (Section 2 ) . Part of this inhibition resides in the binding of the zinc cofactor within the enzyme's active site by captopril's thiol function. Constrained within the active site by the multiple interactions of site and inhibitor, captopril bars entry of angiotensin I and thus prevents its conversion to the most powerful natural pressor, angiotensin 11. Since captopril lacks an amino group, it does not

107

CAPTOPRIL

complex metals in solution with the well-documented avidity (32,33) of amino group bearing thiols like cysteine, glutathione, and in vivo metal depletors like 2 - m e r c a p t o p r o p i o n y ~ g l y c i n e and penicillamine. Indeed, Weiss (30) reported that an alkali potentiometric study of the extent of zinc ion complexation with cysteine (I), 2mercaptopropionyl glycine (11), and captopril (111) indicates the order of binding, at pH 7.4, to be I > I1 > 111. Captopril binds mercuric ion (Section 7 ) to block its colorimetric reaction with Ellman's reagent, thus allowing a measurement of non-sulfhydryl colorimetric interferences. 4.34

Optical Rotation

The optical rotation of the captopril in absolute ethanol (34), using the Perkin-Elmer 155 Automatic Polarimeter, was determined to be: a = -127.8'. The R,S - isomer rotates at about +5'? 4.35 Partition Coefficients A partition ratio (solvent/aqueous) after shaking equal volumes of cosaturated aqueous (pH 2) and methylene chloride was 1.39 ( 2 1 ) . A comparably determined partition ratio between equal volumes of cosaturated 0.1M HC1 and octanol was 1.9 (30). When utilizing salcing out partition from aqueous acid into methylene chloride at the captopril concentrations prevalent in the urine analysis (about 25-50 mcg/ml for a 100 mg dose) NaCl but not Na2S04 was found to enhance captopril oxidation (Section 7). This has been attributed to trace chlorine generation from acidic chloride plus oxygen.

5.

Stability 5.1

Solid State Stabilitv

No significant decomposition was detected (35) in SQ 14,225 bulk samples, stored at +5'c, +33'C and +5OoC f o r up to 6 months or exposed to 900 foot-candles in a light box f o r 30 days, when compared to -2O'C samples which served as the

HAROLD KADIN

108

c o n t r o l . Samples were examined f o r a p p e a r a n c e , c o l o r , o d o r , LD s a f e t y and by q u a n t i t a t i v e TLC and IlPLC , i o d i m 2 p r i c t i t r a t i o n , i n f r a r e d , and optical rotation. 5.2

Solution S t a b i l i t y

C a p t o p r i l i n aqueous s o l u t i o n undergoes an oxygen f a c i l i t a t e d , f i r s t o r d e r , f r e e r a d i c a l oxidation a t its t h i o l to yield captopril d i s u l f i d e ( 2 8 ) . H y d r o l y s i s a t t h e amide l i n k a g e o c c u r s o n l y u n d e r f o r c i n g c o n d i t i o n s (see S e c t i o n 5 . 2 5 ) . O x i d a t i o n w a s d e l a y e d by a d j u s t m e n t t o lower pH, a d d i t i o n o f c h e l a t i n g a g e n t s , i n c r e a s i n g captopril concentration, u t i l i z a t i o n of nitrogen o r l o w oxygen h e a d s p a c e , and i n c o r p o r a t i o n o f a n t i o x i d a n t s . O x i d a t i o n seems t o o c c u r l e s s r e a d i l y i n methanol ( 3 6 ) . N o d e g r a d a t i o n o f c a p t o p r i l w a s o b s e r v e d ( 4 0 mcg/ml) i n t h i s s o l v e n t f o r up t o 2 weeks a t 5OC. 5.21

S t a b i l i t y and S o l u t i o n pH

O x i d a t i o n r a t e c o n s t a n t s a t v a r i o u s pli v a l u e s ( 2 8 ) i n Table 5 , s u g g e s t t h a t c a p t o p r i l i s o p t i m a l l y s t a b l e below pH 3.5, t h e o x i d a t i o n r a t e b e i n g e s s e n t i a l l y c o n s t a n t from pH 2 t o 3. The r a t e c o n s t a n t s i n c r e a s e r a p i d l y above pH 4 . Using HPLC and c o l o r i m e t r y ( 3 8 ) , c a p t o p r i l a q u e o u s s t a b i l i t y w a s s t u d i e d , a t 50 mcg/ml, i n a r o t a t i n g b a s k e t d i s s o l u t i o n a p p a r a t u s f o r up t o 1 8 0 m i n u t e s a t 37OC i n d i s t i l l e d water, and a t pH 1, 2 and 3 . E x c e l l e n t s t a b i l i t y a t pH 1 and 2 b u t a p p r e c i a b l e d e g r a d a t i o n a t pH 3 , and i n d i s t i l l e d w a t e r w a s observed. S u r p r i s i n g l y , t h e r a t e of d e g r a d a t i o n a t pH 3 exceeded t h a t i n d i s t i l l e d water. The more r a p i d o x i d a t i o n a t pH 3 w a s a t t r i b u t e d t o c a t a l y s i s v i a g r e a t e r t r a c e m e t a l s o l u t i o n from the dissolution baskets.

109

CAPTOPRIL

Table 5 Oxidation Rate Constant for Captopril (5 mg/ml) in Citrate-Phosphate Buffers at Various pH Values at 5OoC PH

Rate-Eonstan (day ) x 10f 8.38 9.01 8.22 8.31 9.92 9.13 12.94 19.43 28.93 42.03

2.13 2.59 2.89 3.13 3.53 3.88 4.23 4.67 5.16 5.63 5.22

Solution Stability, Metal Ions and Chelating Agents

Transition metal ions most effectively catalyze oxidation of captopril through a recycling of oxygen free radicals (28). The most effective of these catalysts are ubiquitous copper and iron, in given order. As little as 1 ppm of copper has been observed to catalyze captopril oxidation in solution (28). As has been demonstrated with cysteine (39) lower levels of disodium edetate (EDTA Na2) may enhance metal ion catalyzed thiol oxidation, whereas higher levels retard oxidation. Disodium edetate 0.1% (Na2EDTA 0.1%) best stabilized 0.5 mg captopril per ml (of citrate-phosphate buffer at pH 4, p = 0.5) in Teflon-faced rubber sealed vials (37). Analysis of urinary captopril was necessary for dosage form bioavailability and dose titration studies. The necessity for long term storage of samples prior to analysis required development of an acid-chelate stabilization (40). This stabilization utilized diethylenetriamine pentaacetic acid (DTPA) reputed (40) to be a more effective metal chelator than Na2EDTA. A

HAROLD KADIN

110

relatively large amount of DTPA (about 4 0 0 mg in 5 ml) was mixed into the periodic urine voiding. This was followed by an acidification to about pH 2 with a mixture of citric and oxalic acids and a rapid refrigeration. DTPA, citric and oxalic have been reported ( 4 1 ) to be effective sequestering agents in the pH range 2 to 5 for Al, Cu, Fe, Ni and Zn. Analysis of captopril in urine samples stabilized in this manner, before and after 90-120 days of refrigeration, agreed remarkably well(40). 5.23

Concentration and Solution Stability

The greater the captopril concentration, the slower the oxidation ( 2 8 ) . For example, no significant degradation (within + 3 % ) could be detected by automated Ellman colorimgtry ( 4 2 ) at a concentration of 2 5 0 mg captopril per ml of solution at pH 1 2 . 5 - 1 4 after overnight room temperature storage in uncovered beakers. At the opposite extreme, captopril at an analysis concentration of 2 5 - 5 0 micrograms per ml of solution at pH 1 3 . 5 stored overnight at room temperature in open tubes lost 8 4 % of its sulfhydryl activity ( 2 1 ) . 5.24

Oxygen Tension and Solution Stability

The following 2 1 / 2 hour accelerated stability at 4OoC on 2 5 ml of 1 mg captopril per ml solutions in unstoppered 100 ml volumetric flasks using air, oxygen, or nitrogen purging, where indicated, was carried out ( 2 8 ) . Table 6 Oxygen Tension and Solution Stability M

PH

1 2 3

0.08 0.08

4

0.16 0.16

5 6

0.08 0.04

7.9 7.9 7.5 7.5 7.8 7.8

Code

Na2HP04

(KH2P041

PPm Cu 2 2 10

10

Purge None Air None Air

2

N2

1

O2

8 Captopril Recovered (HPLC) 73 58 88 62 96 0

111

CAPTOPRIL

Under these conditions it is apparent that oxygen causes rapid, complete degradation, air facilitates degradation, and nitrogen protects captopril in solution. 5.25

Amide Hydrolysis and Solution Stability

Captopril solutions at 5 mg per ml in 0 . 5 M HC1, containing 0.1 mg EDTANa /ml to minimize oxidation , were heated at elegated temperatures. The rate of hydrolysis was monitored by HPLC. The data yielded first order linear plots from which Wang (43) calculated rate constants (Table 7). An Arrhenius plot yielded a heat of activation for amide hydrolysis of 21.4 kcal/mole, comparable to other amides. Table 7 Rate Constants for Captopril Hydrolysis

4OoC 4OoC 22oc 22oc

PH PH PH PH

3

4 3 4

k -

t90%

-1 5.5 x lo,*-7 hr-l 5.5 x hrml 6.7 x 10-lOhr -1 6.7 x 10 hr

10 10 86 86

years years years years

It is clear from these t ' s (times for 90% amide hydro1ysis1 , that hydro188f s contributes insignificantly to degradation.

HAROLD KADIN

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6.

Analytical Tests and Methods 6.1

Elemental Analysis

The following results were obtained: Element Calculated C H N S

49.78 6.96 6.45 14.77 6.2

Found for Batch 5 49.97 6.84 6.50 14.63

Spectrophotometric Methods 6.21

Colorimetry

Spectrophotometry for captopril has included the widely utilized Ellman's reaction (44) in which sulfhydryl reduction of 5,5'-dithiobis-2nitrobenzoic acid yields a mole of intensely yellow 2-nitro-5-thiobenzoate anion per mole of captopril. Manual (40,451 and automated colorimetries have been utilized for captopril analysis in chows (Section 7.71), urine (Section 7.72) and in pharmaceutical formulations by TLC (Section 6.31). Kinetic colorimetry vs pH (from pH 5 to 10) studies (46) of a manual version of Ellman's colorimetry of captopril established that the pH optimum was at pH 7 (1 M phosphate, 0.05 M EDTA) Maximum color was attained within 2 min. and stability was maintained for at least 45 min.

.

The S-nitroso-Bratton-Marshall colorimetry (47) has been applied to analysis for captopril in various formulations (48). It was adapted from a method (47) for cysteine in biological materials. In this method the thiol reacts with nitrous acid to form a relatively stable S-nitroso derivative. Excess nitrous acid is destroyed by sulfamic acid. The S-nitroso derivative is then hydrolyzed by mercuric ions to release nitrous acid. The latter diazotizes sulfanilamide, presumably at a faster rate than destruction of the nitrous acid by

CAPTOPRIL

113

excess sulfamic acid. The diazotized sulfanilamide is then coupled to N-(1-naphthy1)ethylenediamine to yield a stable measureable red azo dye. The method was found to be less suitable for analysis of captopril in urine than Ellman's Reaction (Section 7.72). Five simple, spectrophotometric identity tests for captopril reported by Valatin (49) include the observation of an evanescent red when captopril reacts with nitrous acid. In addition captopril color tests were described yielding purple with nitroprusside, blue with ferric chloride, red on addition of neutral N-ethylmaleimide followed by strong alkali, and finally orange-yellow (specific for proline) on acid hydrolysis followed by neutralization then reaction with ninhydrin. 6.22

Fluorometry

Captopril was reacted with N-[p-(2-benzoxazoyl)- phenyl] maleimide in pH 6.85 buffer and the fluorescence of the captopril-maleimide adduct was then measured at 310 nm excitation and 365 nm emission (49). For coupling of captopril to N-(7-dimethylamino-4-methyl coumarinyl) maleimide to yield a highly fluorescent derivative see Section 7.5. 6.3

Chromatosraphic Methods 6.31 Thin Layer Chromatography (TLC)

For quantitation of captopril (purity assay) standard and sample solutions are each chromatographed at 100 pg and 200 pg levels on Analtech silica gel G plates using the solvent system benzene-acetic acid (75:25). The captopril zones on the plate are located by spraying a guide-channel with a basic methanolic solution of 5,5'-dithiobis-2-nitrobenzoic acid. Captopril on the guide-channel appears as a yellow zone. The silica gel containing captopril zones on the untreated portion of the plate are removed from the plate, eluted with 5% aqueous trichloroacetic acid, and reacted with a methanolic solution of 5,5'-dithiobis-2-nitrobenzoic acid, at an alkaline

HAROLD KADIN

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pH, to form an intensely yellow 2-nitro-5thiobenzoate anion that is measured at 412 nm in a spectrophotometer (50)

.

In a thin-layer semi-quantitative procedure for measuring the individual impurities in captopril, samples are chromatographed at 100 pg and 200 pg levels and standards are chromatographed at concentrations ranging from 0.5 pg to 4.5 pg. TLC separation is carried out on Analtech Silica Gel G plates, developed in conventional and in continuous development chambers, using the solvent system benzene-acetic acid (75:25). After development, the plates are air dried and placed in a chamber saturated with iodine vapors. A semi-quantitative estimate of the concentration of each impurity in the sample is made based on a visual comparison of the size and intensity of each impurity zone with the appropriate standard zones (50). These TLC procedures, after appropriate initial extractions, have been adapted for formulations. They have also been applied to semi-quantitative analysis of captopril disulfide in tablets and as a TLC identity test for captopril in tablets (50). Streaked or spotted plates should be introduced into the developing chamber (2 per chamber) immediately after applying the solutions to the plates. This minimizes the conversion of captopril to its disulfide. Placebo powders were spiked with known amounts of captopril and assayed using the TLC procedure. The results of 15 assays gave a recovery of 100.6%, a standard deviation ( s ) of 1.39 and a coefficient of variation (C.V.) of 1.38 (50)

6.32

High-Performance Liquid Chromatography ( HPLC)

Three HPLC systems were investigated (51) for selective separation of captopril from pharmaceutical excipients, synthetic intermediates, degradation products and impurities. These included anion exchange, amino, and

CAPTOPRIL

115

octadecylsilane (ODS) systems. The first and second appeared not to be the systems of choice when they did not achieve necessary separations of pharmaceutical excipients from captopril. A heavily loaded version of the third system ( 1 5 % O D s , monolayer bonded on silica, Partisil ODS-2) was selected as optimum for bulk and tablet analysis. The third system was clearly superior. Nevertheless, the first two systems had very occasional usage when formulations contained excipients which interfered with captopril or its disulfide analysis in the heavily loaded C18 system. The characteristics of these systems and of several others investigated are summarized in Table 8 below. Since captopril has a U.V. absorption maximum at about 2 0 0 nm with broad end absorption (see Figure 1 5 ) U.V. detection was at 230 nm or lower, compatible with a good baseline and low solvent interference inherent in a good signal to noise ratio. An alternative to U.V. detection, for captopril per se (see Section 7.4 for captopril U.V. derivative HPLC) is the electrochemical detector (systems 8 and 9) introduced for sulfhydryl analysis by Saetre and Rabenstein ( 5 2 ) . The electrochemical detector's near neutral detection potential makes it especially thiol selective. It has been reported (53) to have a sensitivity at least 200 fold over that of U.V. detection.

Fig. 15. U.V. Spectrum of C a p t o p r i l in Mobile Phase System 4 (Table 8)

Instrument: Beckman Acta C I11

116

-

Table 8

C a p t o p r i l HPLC S y s t e m s System No.

1

2

Flow Rate ml/min 0.6

1.0

Loop Injection Volume p 1 20

20

S t a t i o n a r y Phase

Mobile Phase

250 x 4 . 6 mm P a r t i s i l SAX 10um S t r o n g a n i o n Whatman

U.V. 5 2 5 mg c i t r i c acid. H20 220 nm +37.7mg N H 4 C I T to 1 liter with CH30H a d j u s t t o p H 3.30 w i t h U.V. 0.1 N HC1 220 nm

300 x 3 . 9 mm u Bondapak NH2 1 0 um a m i n o

Detection

Remarks and Reference 51

51

0 . 0 1 M N a EDTA i n 0.05% SACCH3CN, 9 5 : 5

U.V. 2 2 0 nm

51

CH,OH-H,O-85% ~ ~ $ 20 7 : 7~ 5: '

U.V. 2 2 0 nm

51

Waters 3

1.0

20

250 x 4 . 6 mm P a r t i s i l ODS 5% 10um ODS monol a y e r Whatman

0.1

System No.

Flow Rate ml/min

Loop Injection Volume p 1 Stationary Phase 250 x 4 . 6 mm Partisil ODS 15% lOpm ODS mono layer Whatman

Mobile Phase

Detection

CH OH-H 0-85% H3$04, 3 0 :50: 0.05

U.V. 220 nm

4

1.0

20

5

1.0

20

6

1.0

1.0 (ml)

CH OH-H 0 - 1 8 H3$04, $3 :57 : ODS + trimethyl- 1.0 silylimidazole monolayers Shandon

7

1.0

20

2 5 0 x 4 . 6 mm Partisil ODS 5 % 10pm ODS mono layer Whatman

200

mm Hypersil

+

TSIM 5um + trimethylsilylimidazole monolayers Shandon ODS ODS

H go4, 47.5: 52.5:l. 0

200 mm Hypersil ODS + TSIM 5pm

CH30H-(0.1 M KH PO +

152mMfi2P04) ,

55% :456

U.V. 230 nm

U.V. 230 nm

Remarks and Reference 51 Optimum resolution system

54

54 As say disulfide in low potency captopril formulations

Electrochem., 53 + 0.10 V , Hg Pool vs Ag/AgCl

8

0.5

20

300 x 4.6 mm MC Chromegabond 209 10um heavily loaded ODS ES Industries

CH30H-(0.05 M KH PO + 30mM H $0 53% :15%

Electrochem., 55 + O.lOV, Adapted Hg film for vs. Ag/AgCl "total" urinary captopril analysis

250 x 4.6 mm Partisil ODS 5% 10pm ODS Whatman

CH OH-H 0-85% H3d04, ?0:80: 0.1

U.V. 214 nm

f,

9

1.0

10

1-2

Waters 300 x 3 . 9 mm p Variable Porasil 1Opm Volume silica gel normal phase Waters

CH C1 : HAc, 9 l2

U.V. 240 nm

11

0.5-1

Waters 250 x 4.6 mm Variable Lichrosorb RP18 Volume 5pm "capped" ODS Merck

H O(to pH 3.0 z6.5 M H3P04) - CH3CN, 65 : 35

U.V. 220 nm

12

100

--

c

W

20

ODS = Octadecylsilyl

T

30 x 5.7 cm Prep- CH OH-H20-0.05 PAK C18 ODS M PO 45 : Waters 55 ? 0?65

d

RI

56

57 Qua1 Sepn. of captopril from its R,S isomer

.

58

59 Prep. Sepn. captopril impurities

HAROLD KADIN

120

6.33

Gas Liquid Chromatography (GLC)

A c a p t ~ p r i l - ~synthesis ~c separated captopril from its R,S optical isomer by GLC of its methyl ester (after CH2N2 treatment) on 1.5% OV-17 at 18OOC (60). The gas chromatogram of the pre-resolution isomer mixture showed two peaks of almost the same peak area. After a dicyclohexylamine resolution, GLC of the desired captopril (S,S fraction) indicated a single peak at the retention time of the second peak in the chromatogram of the mixture. The method is recommended, over the more conventional, appreciably less sensitive, measurement of optical rotations (Section 4.34), for process control of the isomer separation. G.C.-mass spectrometry (Section 7.2) and G.C.-flame photometry (Section 7.3) of the N-ethyl maleimide adduct (Section 7 ) of captopril as methyl and heptafluoroisopropyl esters, respectively, have also been reported. 6.4

Titrimetric Methods

Acid iodate (Iodine) titration, before and after chromatography through a Jones Reductor Column was used to measure disulfide in captopril (61). For small amounts of disulfide this technique measures, relatively inaccurately, small differences between two large numbers. Therefore the subsequently developed, more accurate TLC (Section 6.31) and HPLC (Section 6.32) direct assays are preferred. However the iodine titration, with starch indicator, is useful as a thiol purity assay (61). A "Dead Stop" end point indicator (62) makes the titration more amenable to automation. 7.

Analysis in Biological Fluids and Tissues and in Animal Rations

Extreme instability of captopril in biological media necessitated development (63) of an immediate, quantitative thiol derivatization with N- ethylmaleimide (NEM) followed by freezing. Studies (64) with radioactive captopril in whole blood had indicated that a 5 min delay before addition of NEM resulted in a 10% l o s s of

CAPTOPRIL

121

captopril whereas a 30 min delay yielded a 65% loss. The NEM derivative was found (65) to be stable in frozen whole blood or in blood stored at 5OC for at least 3 months: it was unstable in a frozen or 5OC phosphate buffer (pH 6 . 8 5 ) when stored for more than 4 weeks. Antioxidants and metal chelators were ineffective (63) as captopril stabilizers in whole blood. Long term stabilization, at the relatively high concentrations of 1 to 100 mcg per ml prevalent in urine after captopril dosage, has been achieved (40) with metal chelators, acid pH, and 5 O C storage (see Section 5.22). Stability and homogeneity analysis of captopril in animal feeds was required for multiple long range toxicological studies. Despite a high proportion of thiol reactive air, inorganics, and proteins in the feeds, stabilization during the analysis was achieved (46) by an isopropanolic extractant fortified with trichloracetic acid and metal chelators (see Section 7.71). 7.1

Thin Layer Radiochromatography (TLRC)

NEM stabilization allowed TLRC of captopril in whole blood (63). Aliquots of whole blood were analyzed for total radioactivity and NEM- treated aliquots were extracted with methanol. Reconstituted residues of the extracts were applied to silica gel GF plates, developed with chloroform/ethyl acetate/glacial acetic acid (4:5:3) and analyzed for radioactivity associated with captopril and its disulfide by zonal analysis. The limit of detection was about 10 ng captopril/ml of blood. In TLRC analysis of the highly radioactive captopril preparations used for drug metabolism studies the thiol of captopril is particularly susceptible to free radical degradation (Section 4.2). Reaction of thiol across the highly active carbonyl double bond of formaldehyde (incorporated in the mobile phase) protects the radioactive captopril during the chromatography (66).

HAROLD KADIN

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When dilute aqueous solutions of captopril and an excess of formaldehyde are used the product of the reaction has been shown by NMR and IR to be the captopril hemithioacetal.

cp

COOH

The hemithioacetal can be reverted to captopril, for sulfhydryl analysis, by acid, base, or bisulfite. 7.2

Gas Chromatography-Mass Spectroscopy (GC-MS)

NEM-Captopril has been determined by GC-MS in the selected ion mode (67) in whole blood, urine (68), amniotic fluid (691, milk (69), and tissues (68) (liver, kidney, lung, and placenta). For determination in whole blood, proteins are precipitated from vortexed whole blood plus internal standard with freshly prepared 10% metaphosphoric acid. The NEM-captopril is absorbed from 0.45 pM membrane filtrates on purified suction-dried Amberlite XAD-2 resin and eluted with a freshly purified (neutral alumina chromatography) ethyl acetate. The drug is extracted from the ethyl acetate into 5% sodium bicarbonate. After acidification and saturation of the aqueous layer with sodium chloride, the NEM derivative is extracted with ethyl acetate. After evaporation of the solvent extract, samples are methylated for GC-MS. The G.C. was carried out with dried helium gas at 6 ml/min on a 3 % OV-101 column with injector at 28OOC and column temperature programming at 200-280°C at 20°C/min. All glass surfaces had been previously silanized with Sylon-CT. The spectrometer was a modified Electronic Associates Quad 300 quadropole. The samples (1 p l ) were coinjected with 0.5 p 1 of N,O-bis(trimethylsily1) trifluoroacetamide. The latter was used to prolong column life. The peak height intensity

CAPTOPRIL

123

data of the m/z 230 and 248 ions was collected by selected ion monitoring. The limit of detection and the practical detection limit with a 90% confidence limit are 5.5 and 16.5 ng per ml of blood, respectively. The internal standard, an NEM blocked, deuterated or fluorinated derivative of captopril, compensates for extraction and other possible losses. The procedure described (67) could be applied to most of the biological media mentioned with but slight revision. However milk required a greater modification. The high fat content of milk made XAD-2 chromatography unfeasible. Therefore NEM-captopril was instead extracted from sodium chloride saturated milk samples with ethyl acetate. 7.3

Gas Chromatography-Flame Photometric Detection (GC-FPD)

A GC method for captopril in blood and urine utilizing a sulfur selective dedicated FPD has been reported (70). Blood is treated with a s o h . of N-ethylma1eimi.de in phosphate buffer s o h (pH 7.4) and with metaphosphoric acid (10% soln); after centrifugation, the supernatant soln. is extracted with ethyl acetate, and, after further purification, the extract is evaporated, the captopril-N-hexylmaleimide adduct is added as internal standard, and the mixture is treated to convert the two adducts into their hexafluoroisopropyl esters for analysis by g.1.c. at 215O on a glass column (1 m x 3 mm) packed with 2 % of OV-210-yn Gas-Chrom Q (80 to 100 mesh), with N (50 ml min ) as carrier gas and flame-photometric detection. Urine is analysed similarly, but without deproteinisation. The calibration graph (peak-height ratio vs. concn.) is rectilinear for 1 to 5 ug ml- of I.

The sensitivity though not specified appears to be in the microgram range. It is therefore not satisfactory for human blood studies which are in the nanogram range.

HAROLD KADIN

124

7.4

High Performance Liquid Chromatography with U.V. Detection (HPLC-UVD)

An HPLC procedure for captopril in whole blood and urine has been reported (71). Captopril is thiol-blocked with p-bromophenacyl bromide (~BPAB)or N-(4-dimethylamino-3,5-dinitrophenyl) maleimide (DDPM), then the addition products were separated and determined by HPLC on a reversed phase column. Captopril in blood or urine could be derivatized with pBPAB then excess pBPAB removed by hexane extraction. The captopril disulfides in the same samples were then reduced by tributylphosphine to captopril which in turn were thiol-blocked with DDPM. HPLC of the adduct mixture thus allowed a separate analysis of captopril or disulfides, before and after reduction. The method is reported to be sensitive, and precise down to 5 ng per ml of whole blood and 0.1 mcg per ml of urine.

7.5

Spectrofluorometry

The drug, stabilized with dithioerythritol is absorbed on a Brinkmann XAD-2 column from an acidified diluted blood and eluted with pH 6.9 phosphate-30% dimethylformamide buffer. Captopril in the eluate is reacted with N-(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM) to form a fluorescent derivative (Section 6.22). After acidification, the derivative is extracted into toluene. The fluorescence is measured at 375 nm/425 nm-excitation/fluorescence. The analysis was validated by the GC-MS procedure described above in the range of 0.5 mcg to 10 mcg per ml of blood (72). 7.6

Radioimmunoassay (RIA)

Development and application of a simple, highly sensitive RIA for NEM-captopril in blood plasma has been described (89). The lower detection limit is 2 ng per ml of plasma.

125

CAPTOPRIL

7.7

Semiautomated Ellman Colorimetry, (Figure 16) 7.71

Semiautomated Sulfhydryl Analysis in Laboratory Animal Rations

Feeds were extracted with isopropanol fortified with trichloroacetic, ethylenediamine tetraacetic, and diethylenetetramine pentaacetic acids. Pigments in the initial extract and turbidity on aqueous dilution necessitated a cleanup using isooctane after aqueous dilution of the extract. Further aqueous dilution of the isopropanol-water extract insolubilized trace isooctane and more polar lipids. Finally centrifugation in open top tubes removed the last traces of isooctane. In the design of the colorimetry from manual colorimetry ( 7 3 ) the sequence of adding Ellman's reagent before the alkali was reversed to maintain solubility of Ellman's within the autoanalyzer lines. In addition, methanol and aqueous buffer were added to Ellman's reagent to enhance both stability and solubility. Finally the pH of Ellman's reaction was maintained at pH 8.5 rather than at its optimum pH 7 (Section 6 . 2 1 ) to dissolve trace protein haze. Due to the limited selectivity of the colorimetry it was necessary to analyze for and subtract the response of drug- unfortified (blank) feed. The cleanup served to minimize this blank such that captopril could be analyzed down to 0.03% in a variety of animal feeds with good accuracy. 7.72

Semiautomated Ellman Colorimetry for Urinary Captopril and Its Disulfides

The necessity for long term storage of samples prior to analysis and the presence of an oxidation-prone thiol of captopril required development of an acid-chelate stabilization method for urinary captopril (Section 5.22). Thinlayer radiochromatography (Section 7 . 1 ) indicated that human urinary captopril was primarily free (unchanged) and, in almost equal proportion,

HAROLD KADIN

126

disulfide-conjugated with cysteine ( 7 4 ) . Relatively small amounts of captopril disulfide were observed. An electrochemical reduction (52) was utilized to release disulfide-conjugated captopril for thiol colorimetry. Of several rugged reduction cells evaluated, one with a VycorB Disc separating the anode and the mercury pool cathode was preferred. Methylene chloride partitions from acidified salt-saturated urines, before and after reduction, allowed the measurement of free and disulfide-conjugated captopril. The drug partitioned into the solvent whereas the aqueous phase retained acid protonated, amino group-bearing thiols like cysteine. Subsequent solvent evaporation volatilized other potential colorimetric interferences. An automated colorimetry of 25 samples per hour was developed by utilizing relatively thiol selective Ellman's Reagent, 5,5'-Dithio-Bis-(2-Nitrobenzoic Acid) (Sections 6.21 and 7.71). Results were confirmed by an HPLC method with electrochemical detection, developed while this method was in use (42). 7.8

High Performance Liquid Chromatography with Electrochemical Detection (HPLCECD)

-

Captopril in urine has been determined by an HPLC-ECD procedure in which the drug is separated from interferences on a monolayer bonded, heavily loaded (about 20% carbon) octadecylsilane reversephase column with a mobile phase of 55% methanol and 45% aqueous phosphate buffer (55). A commercially available thin layer mercury film detector was used (Section 6.32). The anodic current, resulting from the oxidation of Hg to Hg-captopril complex, was a linear function of captopril concentration. The peak height was reproducible with a relative standard deviation of 0.54% (n=5). Captopril concentration as low as 0.5 mcg per ml of urine can be quantitated. Sample treatment involved simply centrifugation, filtration, and deoxygenation with nitrogen. Recovery of captopril from urine spiked at 2.0 mcg per ml was better than 95%.

CAPTOPRIL

127

TECHNICON AUTOANALYZER FOR ELLMAN COLORIMETRY WASTE

P r RECORDER

IFLOW CELL I

COLOR I M ETER

WASTE

-

FUNCTION

NOMINAL ML/MIN ~~

2 MIN

MIXING c 01LS

YELLOW

HCI WASH

1.2

YELLOW'

CELL

1.2

YELLoW'

J

GREY

4

,TEA-EDTA

1.2

, AIR

1.o

HCI DIL

0.8

ELLMANS

0.8

WASTE * = SOLVAFLEX SOLVENT

RESISTANT TUBING, OTHER TUBING TYGON TEA-EDTA = 1.86% EDTA Nao 2H20 + 2Ooh TRIETHANOLAMINE + 0.02% TWEEN 80

ELLMAN'S =

0.08% ELLMAN'S

IN 50% MEOH (COLD) 0.01 M ACETATE pH 4.7 +

Figure 16

TECHNICON SAMPLER I I WITH 50 2/1 CAM

HAROLD KADIN

128

7.9

High-Performance Liquid Chromatography with Fluorescence Detection (MPLC-FD)

Jarrott et a1 (75) describe an assay for quantitating plasma captopril levels. Blood from patients taking this drug was collected into tubes containing edetate and ascorbic acid, and the plasma was separated by centrifugation. After addition of an internal standard, the plasma was deproteinized and the supernate was adjusted to pII 6.5. N-(l-Pyrene)-maleimide was added to derivatize captopril and an internal standard to fluorescent adducts. These derivatives then were extracted into ethyl acetate-benzene (1:l) and separated from other derivatized thiols by highperformance liquid chromatography. The sensitivity of the assay was 150 pmoles/ml. The HPLCFD uses a mobile phase of methanol-potassium phosphate buffer (5 mM, pH 6.5) (52:48) and the flow rate was 2 ml/min through a radially compressed octadecylsilane 10 cm x 8 mm (i.d.1 column and a spectrofluorometer with a 20 p 1 flow cell. Excitation was at 340 nm with emission taken at 390 nm. 8.

Drug Metabolism

-

Pharmacokinetics

Two features salient to the metabolism and pharmacokinetics of captopril include a) the reversible transformation of its thiol function to dimer and mixed disulfide (74,76-79) and b) the biological stability of its amide linkage (80, Section 3). The following will emphasize these aspects. 8.1

Blood Level Studies

Captopril is absorbed rapidly as indicated by measurable blood levels of the drug 15 min after ingestion (81). These findings are compatible with reports of the onset of antihypertensive activity as soon a s 15 min after a single oral dose (3) in hypertensive patients and with the rapid onset of blockade of angiotensin I-induced increases in blood pressure in healthy subjects (91). Other studies revealed that the captopril metabolites included its dimer disulfide and the mixed disulfides with glutathione, cysteine, and serum albumin (78,811

.

129

CAPTOPRIL

After single oral dosage with 100 mg radioactive captopril, ten healthy subjects attained a mean maximal concentration ( C ) in blood of 800 + 76 ng/ml at a mean time ) of 0.93 + 0.08 hours (8l,82). Methanol extr%%.on, ultrariltration, and dithiothreitol reduction studies (77) coupled with TLRC (Section 7.1) established that about 5 0 % of the total blood radioactivity was unchanged captopril, about 10% dimer disulfide and the remainder other polar metabolites including protein and non-protein mixed disulfides (81,82). Thereafter the captopril fraction of total radioactivity declined rapidly with time whereas the mixed disulfide fraction increased. The curvilinearity of semilogarithmic plots of these captopril blood levels with time indicated that half-life (t 1 , values could not be accurately calculated. #or unchanged captopril levels, curvilinearity may be due to the mixed disulfide fraction acting reversibly to replenish captopril, possibly extending the duration of pharmacologic activity (34,76-79). However plots of total radioactivity allowed calculation of t of 4.3 + 0.2 hours for the 4-12 hour time inter$al and 1g.4 + 2.6 hours Mean for the 12-48 hour time interval (81): concentrations of total radioactivity in blood (expressed as captopril equivalents) were 41 + 7 ng/ml at 24 hours and 20 + 5 ng/ml at 48 fiours after drug administration.

(Fax

Blood level and urinary excretion studies in

6 hypertensive patients, dosed over a 10 day

perigd, indicated that the metabolic disposition of C-captopril was the same at the beginning and end of the study, and was comparable to the disposition in healthy subjects (83). Radiolabeled captopril was administered on days 1 and 10, and blood and urine samples were analyzed for captopril and its metabolites by radiometric assay procedures. Non-radioactive drug (100 mg t.i.d.1 was used for dosage on Days 3 to 9. 8.2

Urinary Excretion Studies

In the fasting state, absorption averaged

7 0 - 7 5 % of an oral dose, as evidenced by human

urinary excretion studies (81,82,84). The presence of food in the gastointestinal tract

HAROLD KADIN

130

reduces absorption of an oral dose by about 35 to 40% (84).

At least 95% of the radioactivity recovered in urine after a 100 mg oral dose of radiolabelled captopril to mildly hypertensive patients was accounted for as captopril and specific urinary metabolites. Captopril and captopril-cysteine mixed disulfide each accounted for about 45% of the urinary radioactivity (about 3 3 % of the dose) and the disulfide dimer accounted for about 5% of the radioactivity in urine (about 4% of the dose) (74) Additional minor urinary metabolites, in rats and dogs, are S-methyl captopril, captopril-S-methyl sulfoxide, N-acetyl cysteine-captopril and glutathione-captopril mixed disulfides (80). These minor metabolites have not, as yet, been reported in man, but appear to be present in monkeys (90).

.

Renal dysfunction, as measured by creatinine clearance, decreased the excretion rate of captopril after a single 100 mg oral dose (85). The authors suggest a method of dosage reduction, based on creatinine clearance measurements, in cases of moderate to severe renal dysfunction. 8.3

Miscellaneous Distribution Studies

Biological stability of the amide function was confirmed by studies (80) on rat dermal collagen; uptake of radioactive proline was considerable, whereas insignificant uptake of captopril labelled in the proline moiety was observed. Whole body radioautography of rats indicated that captopril ( 5 0 mg/kg-intravenously) did not readily enter the central nervous system whereas it readily entered the placenta of pregnant rats (86) Captopril did not readily enter the breast milk of lactating women dosed with captopril at 100 mg t.i.d. for 7 days (87,88). Relatively insignificant milk peak levels were found (4.7 ng/ml)

131

CAPTOPRIL

9.

Acknowledgments

I would like to express my appreciation to individuals who have been very helpful for the contributions indicated: Drs. J. Fried and M. Porubcan - NMR, Drs. Y.J. Wang and T. Prusik Stability, Mr. A. Restivo and Mr. D. Domina Synthesis and Solubility, Drs. A. Cohen and P. Funke - MS and GC-MS, Ms. M. Malley and Dr. J. Gougatas - Single Crystal X-Ray, Mr. F. Dondzila, Mr. S. Perlman and Dr. J. Kirschbaum - HPLC, Mr. H. Roberts - TLC, Mr. R. Poet and Dr. G. Brewer Proof Reading and Manuscript Clarity, Ms. D. Walker - Word Processing, Dr. D. Cushman History, Drs. B. Migdalof and D. McKinstry - Drug Metabolism - Pharmacokinetics. 10.

References

1.

Ondetti, M.A., Williams, N.J., Sabo, E.F., Pluscec, J., Weaver, E.R.; and Kocy, O., Biochemistry, 10, 4033 (1971).

2.

Gavras, H., Brunner, H.R., Laragh, J.H., Sealey, J.E. Gavras, I., and Vukovich, R.A., New Eng. J. Med., 291, 817 (1974).

3.

Case, D.B., Atlas, S.A. Laragh, J.H., Sealey, J.E., Sullivan, P.A., and McKinstry, D . N . , Progr. Cardiovasc. Dis.; 21, 195 (1978).

4.

Cushman, D.W. and Cheung, H.S., Biochem. 20, 1637 (1971). Pharmac., -

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Rubin, B., Laffan, R.J. Kotler, D.G., O'Keefe, E.H., DeMaio, D.A., and Goldberg, 204, 271 M.E., J. Pharmacol. Exp. Therap., (1978).

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Byers, L.D. and Wolfenden, R., Biochem.,

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Cushman, D.W., Cheung, H.S., Sabo, E.F. and Ondetti, M.A., Biochemistry, 16, 5484 (1977).

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HAROLD KADIN

Cushman, D.W., Cheung, H.S., Sabo, E.F., and Ondetti, M.A., Fed. Proc., 38, 2778 (1979). Cushman, D.W., Pluscec, J., Williams, N.J. Weaver, E . R . , Sabo, E.F., Kocy, O., Chueng, H.S., and Ondetti, M.A., Experientia, 29, 1031 (1973).

11.

Ondetti, M.A., Rubin, B., and Cushman, D.W., 196, 441 (1977). Science, -

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New Drug Application No. 18-343 Submitted by E . R . Squibb & Sons Inc., Approved April 6, 1981.

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Levine, T.B., Franciosa, J.A., Cohn, J.N., 62, 35 (1980). Circulation, -

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Ondetti, M.A. and Cushman, D.W., United States Patent 4,105,776, August 8, 1978.

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16.

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17.

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18.

Karchmer , J H. , "Treatise on Anal. Chem. ' I , Part 11; Volume 13, Page 410 (1966), Edited By Kolthoff, I.M. and Elving, P.J., John Wiley Publishers.

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21.

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1976.

1981. 1976.

.

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CAPTOPRIL

133

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Karten, J. and Kabadi, B., Personal Communication, July, 1980.

25.

Malley, M. and Gougatas, J.Z., Personal Communication, May, 1976.

26.

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27.

Weiss, A.L. and Wang, Y.J., Personal Communication, February, 1980.

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29.

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30.

Weiss, A.L. Personal Communication, May, 1976.

31.

Benesch, R. and Benesch, R., J. Am. Chem. 5877 (1955).

SOC. , 77, -

32.

Funae, Y., Toshioka, N., Mita, I., Sugihara, T., Ogura, T., Nakamura, Y., and Kawaguchi, S., Chem. Pharm. Bull., 19, 1623 (1971).

33.

Jocelyn, P.C. , "Biochemistry of the Sulfhydryl Group", Page 344 (19721, Academic Press.

34.

Dondzila, F., Personal Communication, May, 1981.

35.

Shipkowski, E.R., Personal Communication, October, 1980.

36.

Roberts, H.R., Personal Communication, May, 1977.

37.

Timmins, P., Personal Communication, January, 1980.

38.

Poet, R., Personal Communication, May, 1980.

39.

Hanaki, A. and Kamide, H., Chem. Pharm. Bull., 26, 325 (1978).

134

HAROLD KADIN

40.

Kadin, H., Poet, R.B., "Sequential Electrochemical Reduction, Solvent Partition and Automated Thiol Colorimetry for Urinary Captopril and its Disulfides", J. Pharm. Sci., In Press.

41.

Chas Pfizer and Company, New York, "Chelating Agent Usage Calculator".

42.

Kadin, H., Personal Communication, March,

43.

Wang, Y.J., Personal Communication, September, 1979.

44.

Ellman, G.L. and Lysko, H., J. Lab. and Clin. Med., 70, 518 (1967).

45.

Poet, R., Personal Communication, February, 1978, July 1979.

46.

Kadin, H., Personal Communication, October,

47.

Lidell, H. and Saville, B., Analyst, 84, 188

48.

Whigan, D.B., Personal Communication, January, 197 9.

49.

Valatin, P., Personal Communication, July, 1980, Sept., 1980.

50.

Roberts, H., Personal Communication, January, 1978, August, 1979.

51.

Perlman, S. and Kirschbaum, J., J. Chromat., 206, 311 (1981).

52.

1979.

1978.

(1959).

Saetre, R. and Rabenstein, D.L., Anal. Chem.,

50,

276 (1978).

53.

Yeh, P., Abstracts, No. 60, Page 54, Eastern Analytical Symposium, New York, N.Y., November, 198 0.

54.

Tenneson, M.E., Personal Communication, February-March, 1980.

135

CAPTOPRIL

55.

Yeh, P., Personal Communication, October,

56.

Perlman,

57.

Dondzila, F., Personal Communication, May,

58.

Dondzila, F., Personal Communication, May,

59.

Dondzila, F., Personal Communication, December-January, 1980.

60.

Hasegawa, M., Ohyabu, S., Ueda, T., and Yamaguchi, M., J. of Labelled Compounds and 18 I 643 ( 1981). Radiopharmaceuticals, -

61.

Whigan, D.B., Willis, S.L., and Jenkins, E.J., Personal Communication, October, 1976.

62.

Rohmer, M., Personal Communication, February,

63.

Migdalof, B.H., Singhvi, S.M., and Kripalani, K.J., J. Liquid Chromat., 3(6), 857 (1980).

64.

Yeh, P. and Roberts H., Personal Communication, July, 1979.

65.

Ivashkiv, E. and Roberts, H., Personal Communication, July, 1978.

66.

Egli, P., Personal Communication, October,

67.

Funke, P.T., Ivashkiv, E., Malley, M.F., and Cohen, A.I., Anal. Chem., 52, 1086 (1980).

68.

Ivashkiv, E., Personal Communication, March-April, 1980.

69.

1980. 1980.

S.,

Personal Communication, April,

1979. 1981.

1980.

1979.

Ivashkiv, E., Personal Communication, August,

1979.

136

70.

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Matsuki, Y., Fukuhara, K., Ito, T., Ono, H., 188, 177 Ohara, N . , and Yui, T., J. Chromat., (1980).

71.

Kawahara, Y., Hisaoka, M., Yamazaki, Y., Inage, A., and Morioka, T., Chem. Pharm. Bull., - 29(1), - 150 (1981).

72.

Ivashkiv, E., Personal Communication, August,

73.

Kadin, H., Personal Communication, January,

74.

Kripalani, K.J., Meeker, F.S., Dean, A.V., McKinstry, D . N . , and Migdalof, B.H., Fed. 39, 307 (1980). Proceedings, -

75.

Jarrott, B., Anderson, A., Hooper, R. and 70, 665 (1981). Louis, W.J., J. Pharm. Sci., -

76.

Komai, T., Ikeda, T., Kawai, K., Kameyama, E., Shindo, H., J. Pharm. Dyn., 4, 677

1980.

1977.

(1981).

77.

Wong, K.K., Lan, S.J., and Migdalof, B.H., Biochem. Pharmacology, 30, 2643 (1981).

78.

Migdalof, B.H., Wong, K.K., Lan, S.J., Kripalani, K.J. and Singhvi, S.M., Fed. Proc., - --39, 757 (1980).

79.

Shindo, H., Komai, T., Ikeda, T., Kawai, K., Kawamata, W., and Kameyama, E., J. Pharmacobiodyn., 30, S-9 (1980).

80.

Ikeda, T., Komai, T., Kawai, K., and Shindo, 29(5), 1416 (1981). H., Chem. Pharm. Bull., -

81.

Kripalani, K.J., McKinstry, D . N . , Singhvi, S.M., Willard D . A . , Vukovich. R.A. and Migdalof , B. H. , Clin. Pharmacol. and Therap. , 27, -

82.

636 (1980).

McKinstry, D . N . , Singhvi, S.M., Kripalani, K.J., Willard, D.A., and Migdalof, B.H., "Recent Advances in Hypertension Therapy: Captopril", Excerpta Medica, Page 3, (1981), Amsterdam-Oxford-Princeton.

CAPTOPRIL

137

83.

McKinstry, D.N., Kripalani, K.J., Migdaloff, B.H., Willard, D.A., Clin. Pharmacol. & Ther.. 2 7 . 2 7 0 ( 1 9 8 0 ) .

84.

Singhvi, S.M., McKinstry, D.N., Shaw, J.M., Willard, D.A., and Migdalof, B.H., J. Clin. Pharmacol. (in press).

85.

Rommel, A. J. Pierides, A.M., and Heald, A. Clin. Pharmacol. Ther., 27, 282 ( 1 9 8 0 ) .

86.

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129 (1977).

87.

Devlin, R.G. and Fleiss, P.M., Clin. Pharmacol. & Ther., 27, 2 5 0 ( 1 9 ' 8 0 ) .

88.

Devlin, R.G. and Fleiss, P.M., J. Clin 21, 1 1 0 ( 1 9 8 1 ) . Pharmacol. , -

89.

Pearson, F.M., Martin, V.I., Aldujaili, E.A.S., Williams, B.C., and Edwards, C.R.W., 97, 5 2 4 3 , 1 4 8 ( 1 9 8 1 ) . Acta Endocrinologica, -

90.

Migdalof, B.H., Personal Communication, January, 1 9 8 2 .

91.

Ferguson, R.K., Brunner, H.R., Turini, G.A., Gavras, H. and McKinstry, D.N., Lancet, I, 775 I

11.

(1977)

.

Review Coverage Dates

This review summarizes communications up to June 10, 1 9 8 1 . However Sections 2 and 8 are' updated to about January, 1 9 8 2 .

CEFOTAXIME Farid J . Muhtudi and Mahmoud M . A. Hassan

1

2.

3. 4.

5. 6.

7.

8.

140 140 140 142 142 142 142 142 142 142 142 142 152 152 156 156 159 159 159 159 165 166 167

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 ElementalComposition 1.5 Appearance, Color, Odor, and Taste Physical Properties 2.1 Solubility 2.2 Moisture Content 2.3 pH Range 2.4 Optical Rotation 2.5 Spectral Properties Synthesis of Cefotaxime Metabolism Pharmacokinetics Microbiological Activity Methods of Analysis 7.1 Identification Tests 7.2 Non-Aqueous Titration 7.3 Chromatography 7.4 Spectrophotometry 7.5 Microbiological Assay References

Analytical Profiles of Drug Substances Volume I I

139

Copyright 0 1982 by The American Pharmaceutical Association

ISBN &12-260811-9

140

1.

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN Description

1.1.

Nomenclature

1.1.1 Chemical Names (a) (b)

Sodium 7-[2-(2-amino-4-thiazolyl)-2m e t hox y iminoa c e tam i d o ] c epha 1o s po r a n a t e Sodium 3-acetoxymethyl-7-[

2-(2-amino-

4- t h i a z o 1y 1) -2- m e t hox y i m i n o ] a c e t am id0 ] -3-cephem-4-carboxyla

1.1.2

te

.

(c)

(6 R-trans)-3-[ (Acetyloxy) methyl]-7-[ [ (2-amino-4-thiazolyl) (methoxy- imino) a c e t y 11 amino]- 8-0xo-5 - t h ia-1 -azab i c yclo [ 4 . 2 . 0 ] oct-2-ene-2-carboxylic acid monosodium s a l t .

(d)

(6-R, 7R)-7-[ 2-(2-Amino-4-thiazolyl) glyoxylamido] -3-(hydroxy methyl) -8-0x05-thia-1-azabicyclo [ 4 , 2 , 0 ] oc t-2-ene2-carboxylic a c i d a-(O-methyloxime), a c e t a t e ( e s t e r ) monosodium s a l t .

(e)

5-Thia-1-azabicyclo [ 4.2.01 oc t-2-ene 2-2 c a r b o x y l i c a c i d , 3 - [ ( a c e t y l o x y ) methyl] -7-[ [ (2-amino-4-thiazolyl) (me thox y i m i n o ) y a c e t y l amino ] -8 -0xo- , [6R-[6a,78(Z)II-

Generic Name Cefotaxime sodium; HR 756; RU-24,662; RU-24,756

1.1.3

P r o p r i e t a r y Names C l a f o r a n ; Primafen; Z a r i v i z ; T a r i v i d .

1.2.

Formula

1 . 2 . 1 Empirical

C16H16N507S2Na

.

1.2.2 Structural

m ?2

I

V I

0 =. v 0 I hl X

V

142

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN 1 . 2 . 3 CAS no.

[ 611846-23-31 (1) [ 60846-21-11 (1) [63527-52-61 1.3.

C16H16N507S2Na C H N 0 S (as f r e e acid) 16 1 7 5 7 2

(2)

M o l e c u l a r Weight 477.23

1.4.

E l e m e n t a l Composition C, 40.23;;

H , 3.38%; N , 14.67%;

0, 23.47%; S , 13.44%; Ma 4.82%.

1.5.

Appearance, C o l o r , Odor and T a s t e : White t o creamy w h i t e c r y s t a l l i n e powder, o d o r l e s s and h a s a s a l t y t a s t e a t the b e g i n n i n g , f o l l o w e d by b i t t e r n e s s

.

2.

Physical Properties

2.1.

Solubility F r e e l y s o l u b l e i n water ( 0 . 5 g s o l u b l e i n 5 ml) ( 3 ) , s l i g h t l y s o l u b l e i n alcohol ( a b s o l u t e , 95%), i n s o l u b l e i n chloroform (4)

2.2. M o i s t u r e C o n t e n t Not more t h a n 6 p e r c e n t , d e t e r m i n e d by t h e Karl F i s c h e r method, u s i n g a 0.2 g sample d i s s o l v e d i n 2 m l methanol ( 3 ) . 0

Loss on d r y i n g a t 60 C u n d e r vacuum i n t h e p r e s e n c e o f phosphorus p e n t o x i d e f o r 4 h r . s h o u l d n o t exceed 6 % ( 3 ) . 2.3. pH r a n g e The pH of c e f o t a x i m e a s a 10%a q u e o u s s o l u t i o n i s 4 . 5 t o 6.5 d e t e r m i n e d p o t e n t i o m e t r i c a l l y ( 3 ) . 2.4. O p t i c a l R o t a t i o n

[ a]D ( c = l % aqueous s o l u t i o n ) (on d r i e d b ases) (3).

+

58 t o

+

64

0

The o p t i c a l r o t a t i o n of c e f o t a x i m e ( c = l % aqueous s o l u t i o n ) was d e t e r m i n e d u s i n g a P e r k i n E l m e r 2 4 1 MC P o l a r m a t i c and found t o be:

[ a~D240

+

59.30.

143

CEFOTAXIME 2.5.

Spectral Properties 2.5.1

Ultraviolet Spectra The UV s p e c t r a o f c e f o t a x h e i n a n aqueous s o l u t i o n and i n 0 . 1 N H C 1 a r e p r e s e n t e d i n Fig. 1 and F i g . 2 r e s p e c t i v e l y . These were scanned from 200 t o 400 nm u s i n g a PyeUnicumSP 8-100 U l t r a v i o l e t s p e c t r o photometer. The f o l l o w i n g t a b l e 1 shows t h e UV d a t a .

Table 1.

UV c h a r a c t e r i s t i c s of cefotaxime

c onc en t r a t i o n

solvent

water

0.5 mg/ml 0.05 mg/ml 0.01 mg/ml

0.1N R C l 0.1N HC1

2.5.2

.

E (lX, lcm)

max nm 234 205,262 263

420 (5)

Infrared Spectra The I R s p e c t r a of c e f o t a x i m e a s KBr-disc and a s n u j o l mull a r e shown i n F i g . 3 and The KBr-disc w a s Fig. 4 r e s p e c t i v e l y . recorded on a P e r k i n E l m e r 58OB i n f r a r e d s p e c t r o m e t e r . The s t r u c t u r a l a s s i g n m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s (Table 2).

Table 2.

I R c h a r a c t e r i s t i c s of c e f o t a x i m e

Frequency Cm

-1

Assignment

3420

-NH2

3340 (broad)

-NH,

2940

-S-CH2

1 7 60

-C=O

-NH2

lactam

0 II

1730

-C=O

e a r b o x y l i c , O-C-CH3

1650

0 -C-NH

1620

0 -C-NH,-C=N-,-C=C-

1540

0 -C-N-

1385-13 55

-0-CO-CH3

1180 1050

C=O i n e s t e r

11

C-0

stretching

230 240 250

304

350

400

Fig.1

The UV spectrum of

Cefotaxtme

in

water

200

210

250 2 60 270

300

350

400

Fig.2 The UV spectrum o f C e f o t a x i m e in O . I N H C I .

144

3.0

4000

40

5.0

2500

2000

MICRONS

70

6.0

I

3500

3000

1800

1600

1400

8.0

1200

9.0

10

1000

12

1)oo

14

8 CD 0 0

a0

0 0 0 4

2

0 0

0

$

0

0

0

'0, 0

0

2 0

cu

0

0

0 v)

0

cy

a 0

0

c3

0 0

In rn

CEFOTAXIME

147

2.5.3

Nuclear Magnetic Resonance S p e c t r a

2.5.3.1

Proton SDectra

The PMR s p e c t r a of c e f o t a x i m e i n d e u t e r a t e d d i m e t h y l s u l f o x i d e (DMSOD6) and d e u t e r i u m o x i d e w e r e r e c o r d e d on a v a r i a n T-60A, 60-MHZ NMR s p e c t r o m e t e r u s i n g sodium -2, 2-dimethyl-2-silapentane-5-sulfonate (DSS) a s a n i n t e r n a l r e f e r e n c e . These a r e shown i n F i g . 5 and F i g . 6 r e s p e c t i v e l y . The f o l l o w i n g s t r u c t u r e a s s i g n m e n t s h a v e been made ( T a b l e 3 ) ( 6 ) . T a b l e 3.

PMR c h a r a c t e r i s t i c s of c e f o t a x i m e

Group

Chemical Sh DMSQD6

Et ( P P ) D2°

-0co CH,

2.00(s)

2.12(s)

2-G2

3 . 3 3 (9)

3 . 5 3 (9)

N - E 3

3 . 8 3 (s)

4.00(s)

4.97(9)

4.83 ( d )

4.97(q)

5.20(d)

7-H

5.60(2d)

5.82 ( d )

5-H

6.70(s)

6.97 (s)

2-Nl12

7.22 (bs)

CONH -

9.47 ( d )

0-CH -3 6-H

AC

.

-

s = s i n g l e t , d=doublet, q = q u a r t e t , 2d=doublet of d o u b l e t s , bs=broad s i n g l e t . 13 2.5.3.2 C-NMR 13 C-NMR c o m p l e t e l y d e c o u p l e d and o f f r e s o n a n c e s p e c t r a a r e p r e s e n t e d i n F i g . 7 and F i g . 8 respectively. Both were r e c o r d e d o v e r 5000 H, r a n g e i n d e u t e r i u m o x i d e ( c o n c . 100 m g / l m l D20)

148

0

149

FARID J. MUHTADI AND MAHMOUD M . A. HASSAN

150

4

a

1 I

-J

-TTJ_LLl-

F i g . 7. spectrum

.

'_J _ ' I ' -I

'

' '-11

I

rI ' I '

The 1 3 C NMR of Cefotaxime, completely decoupled

'I I

I

II

Fig. 8 . The 1 3 C NMR of Cefotaxime, o f f resonance (Proton coupled) spectrum

151

CEFOTAXIME

on FT-80A-8OMHz NMF. s p e c t r o m e t e r , u s i n g 1 0 mm sample t u b e and d i o x a n e a s a r e f e r e n c e s t a n d a r d a t 2OoC. T h e c a r b o n chemical s h i f t a r e a s s i g n e d on t h e b a s i s of t h e a d d i t i v i t y p r i n c i p a l s and t h e o f f r e s o n a n c e s p l i t t i n g p a t t e r n ( T a b l e 4) ( 6 ) . 12 NOCH3

!?1 6 OCCH3 15

H2N

COONa

13

T a b l e 4. Carbon no. 8-C=0

Carbon Chemical s h i f t s o f c e f o t a x i m e Chemical s h i f t [ PPm 1 174.69(s)

Carbon no. C-3

Chemical s h i f t [ PPm 1 117.37 ( S )

1o-c=0

171.25(s)

C-5

113.64 (d)

13-C=0

168.78 ( s )

c-7

65.07 (d)

15-C=0

165.08( s)

C-6

59.59(d)

c-2

164.65(s)

C-14

58.23 ( t )

c-11

148.62 (s)

c-2

26.35( t )

c-4

141.40( s)

C-12

63.55 ( 4 )

c-4

132.42 (s)

C-16

21.22 ( 4 )

,

,

s=singlet, d=doublet, t = t r i p l e t , q=quartet.

FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

152

3.

S y n t h e s i s of Cefotaxime Cefotaxime i s a s e m i - s y n t h e t i c c e p h a l o s p o r i n . The s t a r t i n g material f o r s u c h c e p h a l o s p o r i n s i s 7-aminocepha l o s p o r a n i c a c i d (7-ACA) which o b t a i n e d e i t h e r by mild a c i d o r enzymatic h y d r o l y s i s of c e p h a l o s p o r i n C . ( 7 , 8 , 9 , 1 0 )

P r e p a r a t i o n of t h e S i d e Chain:

(5)

The s t a r t i n g m a t e r i a l e t h y l a c e t o a c e t a t e [ l ] w a s o x i mated t o produce oximinoethylacetoacetate [ 21. Methylat i o n of [ 21 followed by bromination y i e l d e d syn-methoxyiminobromoketone [ 3 ] . Condensation of [ 3 ] w i t h t h i o u r e a [ 4 ] i n aqueous medium and a t a low t e m p e r a t u r e gave syna m i n o t h i a z o l y l d e r i v a t i v e [ 51. The a m i n o t h i a z o l y l r i n g w a s p r o t e c t e d by t r i t y l a t i o n t o g i v e t h e N - t r i t y l d e r i v a t i v e [6]. S a p o n i f i c a t i o n of t h e l a t t e r by b o i l i n g w i t h NaOH s o l u t i o n a f f o r d e d t h e c o r r e s p o n d i n g a c i d [ 7 ] . Acyl a t i o n of t h e amino group of 7-ACA w i t h t h e r e s u l t i n g a c i d [ 7 ] proved d i f f i c u l t . T h i s h a s been overcome by t h e u s e of symmetrical a n h y d r i d e [ 8 ] , which w a s o b t a i n e d by condensing two m o l e c u l e s of [ 7 ] i n t h e p r e s e n c e of d i c y c l o h e x y l c a r b o d i i m i d e [ a ] . 7-ACA w a s a c y l a t e d by compound [8] t o g i v e [ 91. Removal of t h e t r i t y l r e s i d u e under a c i d i c c o n d i t i o n gave t h e f r e e a c i d form (R=H) of c e f o taxime [ l o ] . P r e p a r a t i o n of a S t a b l e Sodium S a l t : T h i s w a s prepared by adding t h e s o l i d f r e e a c i d t o a n aqueous a l c o h o l i c s o l u t i o n of a n o r g a n i c sodium s a l t t o g i v e t h e s t a b l e a c t i v e D-form. (5) The s y n t h e s i s of c e f o t a x i m e i s p r e s e n t e d i n Scheme 1. I t i s i n t e r e s t i n g t o n o t e t h a t t h e syn-isomer of cefotaxime i s u p t o 100 times more a c t i v e a g a i n s t c e r t a i n organisms t h a n t h e a n t i - i s o m e r .

4.

Metabolism The metabolism of c e f o t a x i m e i n r a t , dog and man u s i n g 14C-labelled c e f o t a x i m e h a s been s t u d i e d by Chamberlain e t a l . (11). Cefotaxime i s w e l l absorbed i n t h e t h r e e species a f t e r intramuscular administration. It i s e l i m i n a t e d mainly v i a t h e u r i n e . The major m e t a b o l i t e being d e s a c e t y l c e f o t a x i m e . The amount of unchanged c e f o -

153

CEFOTAXIME OH

Scheme 1:

S y n t h e s i s of Cefotaxime

N’

I

methylation bromination

then

N,0CH3

+

H2N
‘OC2H5

S

/

CH*

Br

[41

condensation

H2N

i

[31

-(syl:*5 s

[ 51 tr i t y l a t ion

154

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

condensation C 6H 1 1 N=C=NC6H1

[a1

H C-0 3

/

N

+

symmetrical anhydride

[81

COOH

1

7- A M

Acylation

155

CEFOTAXIME

0-CH


Tr,n

H

3

/

(4 CH 20Ac

0'

S

C02H

1

acidification

0 -CH N'

H 2N

3

~ R=H acid R=Na Cefotaxime

C02R

[lo1

~ cH20Ac

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

156

taxime and t h e major m e t a b o l i t e e l i m i n a t e d i n t h e u r i n e i s s i m i l a r f o r each s p e c i e s . Two f u r t h e r m e t a b o l i t e s , UP1 and UP2 a r e d e t e c t e d i n dog and human u r i n e b u t n o t i n r a t urine. The f o r m a t i o n of b o t h UP m e t a b o l i t e s i s r e l a t e d t o t h e r a t e of r e n a l e l i m i n a t i o n of d e s a c e t y l c e f o t a x i m e ( 11) * The m e t a b o l i c pathway f o l l o w s t h e r o u t e i n dog and human which may occur i n t h e l i v e r i s p r e s e n t e d i n Scheme ?

L.

5.

P harmacokine t i c s The p h a r m a c o k i n e t i c s of c e f o t a x i m e w a s r e p o r t e d t o be d o s e independent f o r d o s e s up t o 2.0 g ( 1 2 ) . Following t h e i n t r a m u s c u l a r (i.m.) i n j e c t i o n of a 0 . 5 g (13) and 1 . 0 g d o s e s ( 1 2 ) , t h e mean peak serum l e v e l of 11.7 \ig/ml and 22 ug/ml a t 0.5 h were o b t a i n e d r e s p e c t i v e l y . A f t e r i n t r a v e n o u s ( i . v . ) p o l u s i n j e c t i o n of 1 . 0 g c e f o t a x i m e , t h e mean peak l e v e l w a s 105 ug/ml and an a p p a r e n t volume of d i s t r i b u t i o n of 25 l i t r e s w a s e s t i m a t e d ( 1 2 ) . Values of 32 and 37 l i t r e s f o r t h e volume of d i s t r i b u t i o n were a l s o r e p o r t e d ( 1 3 ) . Cefotaxime i s mainly e l i m i n a t e d by r e n a l e x c r e t i o n and s e r u m - h a l f - l i f e ranged from 0.92 t o 1.35 h a f t e r 1 g -i.m. i n j e c t i o n , and from 0.84 t o 1 . 2 5 h a f t e r i . v . a d m i n i s t r a t i o n ( 1 2 ) . The m a j o r i t y of t h e d o s e being e x c r e t e d unchanged i n t h e u r i n e (-51% w i t h i n 8 h) (12). I n a n o t h e r s t u d y (13) t h e serum c l e a r a n c e of 341 mlfmin. per 1.73 m2 and r e n a l c l e a r a n c e of 130 mlfmin p e r 1.73 m2 were o b t a i n e d . P r o t e i n b i n d i n g of c e f o t a x i m e ranged from 35 t o 45%.

6.

Microbiological Activity Cefotaxime p o s s e s s e s a n e x t r e m e l y broad a n t i b a c t e r i a l C positive bacteria spectrum and i s v e r y a c t i v e a g a i n s t e s p e c i a l l y on E n t e r o b a c t e r i a c e a e , Haemophilus i n f l u e n z a e o r Neisseria gonorrhoeae. I t is a l s o a c t i v e on Bacteroides f r a g i l i s and Pseudomonas a e r a g i n o s a ( 1 4 ) . Cefotaxime p e n e t r a t e s w e l l through t h e c e l l w a l l s , showing h i g h 6l a c t a m a s e s t a b i l i t y and s t r o n g a f f i n i t y t o t h e i m p o r t a n t t a r g e t enzymes ( 1 4 ) .

CEFOTAXIME

157

Scheme 2 :

Metabolism of Cefotaxime. OCH3 N'

coCefotaxime Deacetylation

'N

2

OCH3

-<: D;k"oE(A co-

NH Desacetyl

CH OH

cefotaxime

ELo 9 0

Desacetyl cef o t a x ime l a c t o n e

0

FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

158

N’

OCH3

+

UP 2

H

H

CEFOTAXIME

7.

159

Methods of A n a l y s i s

7 . 1 . I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s are mentioned under c e p h a l e x i n i n t h e B.P. 1980

(15) :A) The i n f r a - r e d a b s o r p t i o n spectrum determined i n t h e s o l i d s t a t e , i s concordant w i t h t h e r e f e r e n c e spectrum of c e f o t a x i m e . B) Mix 20 mg w i t h a few d r o p s of an 8 0 % V/V s o l u t i o n of s u l f u r i c a c i d c o n t a i n i n g 1 % V/V of n i t r i c a c i d ; a yellow c o l o r is produced. C) Mix 20 mg w i t h 5 d r o p s of a 1 % V/V s o l u t i o n of g l a c i a l a c e t i c a c i d and add 2 d r o p s of a 1 % W/V s o l u t i o n of copper s u l f a t e and d r o p of 2M sodium hydroxide; an o l i v e - g r e e n c o l o r i s produced.

7.2.

Non-Aqueous T i t r a t i o n Assay Weigh a c c u r a t e l y a b o u t 0.15 g of c e f o t a x i m e and d i s s o l v e i n 50 m l g l a c i a l a c e t i c a c i d . Assay potentiometrically with 0.1 N perchloric acid.

1 m l of 0.1 N p e r c h l o r i c a c i d c o r r e s p o n d s t o 23.87 mg of c e f o t a x i m e ( 3 )

.

7.3.

Chromatography

7.3.1

Thin Layer Chromatography (TIX) TLC of c e f o t a x i m e i s perforlned on s i l i c a g e l c o a t e d , s e l f - i n d i c a t i n g chroma t o g r a p h i c p l a t e s (60 F 2 5 4 , Merck).

The developing s o l v e n t system is:-

Ethylacetate/Ethanol/Water/Formic a c i d (60 : 25 : 1 5 : 1) by volume. Development i s c a r r i e d o u t i n a l i n e d t a n k , and t h e s o l v e n t i s allowed t o m i g r a t e a b o u t 1 5 cm from t h e s t a r t i n g p o i n t s . The p l a t e s are v i s u l i z e d by examining under u l t r a v i o l e t l i g h t a t 254 nm ( 3 ) .

FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

160

The above method c a n be adopted f o r t h e d e t e r m i n a t i o n of t o t a l i m p u r i t y c o n t e n t of cefotaxime as f o l l o w s ( 3 ) :A 2 % s o l u t i o n of t h e sample t o be determined i s d i s s o l v e d i n a m i x t u r e of 8 v o l . a c e t o n e and 2 v o l . water. Cefotaxime r e f e r e n c e s u b s t a n c e (HR 756 s t a n d a r d ) i s d i s s o l v e d i n t h e same m i x t u r e t o produce 0.02 %, 0.04 % and 0.08 % s o l u t i o n s .

Two s p o t s 0.01 and .0.02 m l of t h e t e s t e d s o l u t i o n s are a p p l i e d t o t h e c h r o m a t o p l a t e t h e s e r e p r e s e n t i n g 200 and 400 pg r e s p e c t i v e l y . A 0.01 m l of e a c h s t a n d a r d s o l u t i o n is a l s o applied t o t h e chromatoplate representing 2 , 4 , 8 p g r e s p e c t i v e l y . The p l a t e i s developed a s above. The t o t a l i m p u r i t y c o n t e n t should n o t exceed 5 %.

Another TLC system i s recommended f o r cefotaxime a s f o l l o w s : A s o l u t i o n of c e f o t a x i m e i s a p p l i e d i n t o a s i l i c a g e l G c h r o m a t o p l a t e . The p l a t e i s developed i n t h e s o l v e n t Methanol : Ammonia (100 : 1 . 5 ) . A f t e r development, t h e p l a t e i s a i r d r i e d , sprayed w i t h 0.5 % i o d i n e i n chloroform. Cefotaxime developed a d a r k brown s p o t which has Rf v a l u e of 0.83 ( 4 )

7.3.2

Paper Chromatography Descending paper chromatography w a s accomplished u s i n g Whatman no. 1 paper. The s o l v e n t system c o n s i s t e d of i s o p r o p a n o l p y r i d i n e - w a t e r (65 : 5 : 30, V / V ) . The chromatogram was s u b j e c t e d over-night f o r development. Examination was conducted under t h e UV l i g h t a t 254 nm.

7.3.3

Gas Liquid Chromatography (GLC) A GLC method f o r t h e d e t e r m i n a t i o n of cefotaxime h a s been adopted i n our l a b o r a t o r y

CEFOTAXIME

161

u s i n g a Varian GC - 3700 g a s chromatograph equipped w i t h Varian CDS 111 i n t e g r a t o r . Column c o n d i t i o n s : 3 Z OV-17on chromosorb WHP (80 - 100 mesh) g l a s s column (2 m x 2 mm) The column w a s c o n d i t i o n e d i s o t h e r m a l l y a t 120° f o r 1 0 min. and t h e n t h e tempe r a t u r e w a s programmed a t 10°/min up t o 2100. C a r r i e r g a s : Helium, f l o w r a t e was a d j u s t e d t o 20 m l / m i n . D e t e c t o r : FID, hydrogen and a i r f l o w rates were a d j u s t e d t o 40 ml/min. and 300 ml/min. respectively Ethanol w a s used a s t h e s o l v e n t and t h e c h a r t speed w a s a d j u s t e d t o g i v e 0.5 cm./min. The r e t e n t i o n t i m e = 8.15 min. The GLC of cefotaxime i s p r e s e n t e d i n F i g . 9.

.

Another GLC method h a s been d e s c r i b e d € o r t h e d e t e r m i n a t i o n of r e s i d u a l s o l v e n t s i n cefotaxime ( 3 ) . Apparatus :

A 1 . 5 m PORAPAK Q column programed a t 150' with a flame i o n i z a t i o n d e t e c t o r was used. I s o p r o p a n o l w a s used a s t h e i n t e r n a l s t a n d a r d .

Solution I :

70 mg cefotaxime s t a n d a r d (HR 756) and 0.08 mg i s o p r o p a n o l were d i s s o l v e d i n water t o produce 1ml.

S o l u t i o n I1 : 0.05 mg methanol, 0.13 mg e t h a n o l and 0.08 mg i s o p r o p a n o l were mixed w i t h water t o produce 1ml. Procedure :

S o l u t i o n s I and I1 w e r e i n j e c t e d and t h e s o l v e n t l e v e l s w e r e determined from t h e peak h e i g h t s c o r r e c t e d a g a i n s t t h e i n t e r n a l standard.

Results :

Methanol should n o t exceed 0.2 % e t h a n o l o r any o t h e r o r g a n i c s o l v e n t should n o t exceed 1 % .

FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

162

F i g 9.

GLC of c e f o t a x i m e

A = cefotaxime

7.3.4

High Performance L i q u i d Chromatography (HPLC) S e v e r a l HPLC s y s t e m s f o r t h e i d e n t i f i c a t i o n and s e p a r a t i o n of c e f o t a x i m e and i t s metabol i t e s have been r e p o r t e d . These s y s t e m s a r e g i v e n i n T a b l e 5. HPLC o f c e f o t a x i m e and i t s m e t a b o l i t e s u s i n g system 1 (11) i s p r e s e n t e d i n F i g . 10.

T a b l e 5. System no.

Column

HPLC Systems of Cefotaxime

Mobile P h a s e

1.

A n u c l e o s ill ODS column

Acetonitrilewa t e r - a c e t i c a c i d ( 8 : 9 1 : 1 by v o l )

2.

A Microbondapak,

wa t er-me t h a n o l (39 : 7 ) c o n t a i n i n g 0.005 M h e p t a n e sulfonic acid

C18

corasil

guard column 3.

Si-C18,

10

microns

Methanol-water(1 : 5) KH2 P O 0.06 % 4 N a 2 HPO 2H20

Retention Time (min)

Detect ion ( nm)

13.0

W region

30 .O

254 nm

-

235 nm

Ref.

4 t o r e a c h pH 7 . 8

4.

Li ch r os o r b RP-8

Phosphoric acidmethanol

17.0

310 nm

cL7 1

Table 5 contd.

System no.

5.

Column A Rever sed-phase Hibar RT 250-4 L i c h r o s o r b RP

18-7 um, c o r a s i l C18 37-50 Vm

Mobile Phase 20 m m o l sodium

R e t e n t i o n Time (min)

4.8

Detection

Ref.

(nm) 254 nm

d i h y d r o g enp ho s pha t e

i n water-methanolacetonitr ile

(83:7:10, V / V / V )

guard column

6.

A 10-cm octade-

c y l s i l a n e column a n d a 4-cm precolumn c o n t a i n i n g copellicular oc t a d e c y l s i l a n e

1 4 % methanol, 1% acetic acid, in distilled water

12.0

254 nm ( s e t a t 0.005 absorbance unit)

(19)

CEFOTAXIME

165

A I

D

13 R e t e n t i o n time

(min)

F i g . 1 0 HPLC of C e f o t a x i m e and i t s M e t a b o l i t e s A , D e s a c e t y l c e f o t a x i m e ; B , UP1; C , UP2; D, Desacetyl cefotaxime l a c t o n e ; E , cefotaxime. 7.4

Spectrophotometry A PMR method f o r q u a n t i t a t i v e d e t e r m i n a t i o n of c e f o t a x i m e and o t h e r c e p h a l o s p o r i n s i n b u l k d r u g s and v a r i o u s d o s a g e f o r m s i s r e p o r t e d ( 2 0 ) . The d e t e r m i n a t i o n i s based on t h e i n t e g r a t i o n of t h e 6-H and 1 o r 7-H of t h e @-lactam r i n g system r e l a t i v e t o t h a t of t h e n i n e p r o t o n s of t-butanol. The method i s r a p i d , a c c u r a t e and precise, with an average standard deviations of 2 1 . 5 1 i n s t a n d a r d m i x t u r e s and k 1 . 1 5 i n p h a r m a c e u t i c a l f o r m u l a t i o n s . The p r o c e d u r e f u r n i s h e s a s p e c i f i c means o f i d e n t i f i c a t i o n of e a c h i n d i v i d u a l c e p h a l o s p o r i n a s w e l l a s d e t e c t i o n of t h e commonly e n c o u n t e r e d i m p u r i t i e s .

Procedure: Weigh a c c u r a t e l y a p o r t i o n of t h e powder e q u i v a l e n t t o 100 mg of t h e c e p h a l o s p o r i n i n t o a g l a s s s t o p p e r e d sample t u b e . Add 1.0 m l a c c u r a t e l y measured D20 c o n t a i n i n g a n a c c u r a t e w e i g h t of t - b u t a n o l , s t o p p e r and s h a k e f o r 3 min. T r a n s f e r a b o u t 0 . 5 m l of t h e r e s u l t i n g s o l u t i o n i n t o a NMR t u b e and r e c o r d t h e s p e c t r u m , a d j u s t i n g t h e s p i n r a t e t o r e d u c e t h e s p i n n i n g s i d e bands a s

FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

166

much a s p o s s i b l e . I n t e g r a t e t h e peaks of i n t e r e s t (The 6- o r 7-H of t h e B-lactam r i n g a p p e a r i n g a t 4.86 - 5.80 ppm and t h e 9 p r o t o n s of t - b u t a n o l a p p e a r i n g a t 1.23 ppm) a t l e a s t t h r e e times and determine t h e average i n t e g r a l s . The amount of c e p h a l o s p o r i n i s t h e n c a l c u l a t e d as follows:-

Where Ac i s t h e i n t e g r a l v a l u e of t h e c e p h a l o s p o r i n signal,

%

t h a t of t h e t - b u t a n o l

s f g n a l , E.Wc is t h e

is one b n i n t h of t h e m o l e c u l a r weight of t - b u t a n o l (= 8 . 2 4 ) .

m o l e c u l a r weight of c e p h a l o s p o r i n and E.W

7.5

M i c r o b i o l o g i c a l Assay C e f otaxime i s m i c r o b i o l o g i c a l l y assayed by t h e a g a r w e l l d i f f u s i o n method of Grove and R a n d a l l (21) a s modified by Chabbert and Boulinger ( 2 2 ) .

The f o l l o w i n g t a b l e 6 summarises t h e media and t h e t e s t organisms a s r e p o r t e d . T a b l e 6.

Nedia and Test Organisms Used

Med iurn

1. Agar a n t i b i o t i c no. 2 (Difco) 2.

II

11

3.

II

11

4.

II

II

5.

11

It

6.

I1

II

7 . A n t i b i o t i c no. 3 (Oxoid) 8. Mueller-Hinton agar

Test Organism

Ref.

Spores of B a c i l l u s s u b t i l i s

(23)

B a c i l l u s s u b t i l i s (ATCC 6633)

(24)

S a r c i n a l e u t e a (ATCC 9341)

(24)

Escherichia

coli 3989

(13)

E. c o l i (ATCC 9637)

(26)

Proteus morganii

(19)

Staphylococcus epidermid i s Q 305

(25)

P r o t e u s m i r a b i l i s (ATCC 14273)

(18)

Pseudomonas a e r u g i n o s a ( K 1118)

CEFOTAXIME

167

8.

References

1.

"Annual Drug Data Report", Ed. J . R . P r o u s , V 0 1 . 1 1 1 , J . R . P r o u s S . A . , B a r c e l o n a , S p a i n , p. 5 3 , ( 1 9 8 1 ) .

2.

Chemical A b s t r a c t , I n d e x Guide S u p p l . , 1977-1979 c u m u l a t i v e , The American Chemical S o c i e t y , U.S.A.

3.

C l a f o r a n " Q u a n t i t a t i v e and Q u a l i t a t i v e methods of analysis" Roussel Uclaf, P a r i s , France.

4.

S . Khawaja, U n i v e r s i t y of Riyadh, S a u d i A r a b i a

" P e r s o n a l Communication''.

5.

R. Bucourt, D. Bormann, R. Heymes and ?I. P e r r o n n e t J . A n t i m i c r o b i a l Chemotherapy, 6 , S u p p l . A , p. 6 3 , (1980).

6.

M.M.A.

7.

Hassan and F . J . Muhtadi, "Unpublished Data".

B. Loder, G.G.F. Newton and E.P. 2,4 0 8 , (1961).

Abraham, Biochem.

J.,

8.

R.B. Morin, B . G . J a c k s o n , E . H . F l y n n , and R.W. J . Am. Chem. SOC., 8 4 , 3400, ( 1 9 6 2 ) .

9.

B. F e c h t i g , H. P e t e r , H. B i c k e l and E . V i s c h e r , Helv. Chim A c t a , 2, 1108 (1968).

Roeske,

10. E . H . F l y n n , Ed., C e p h a l o s p o r i n s and P e n i c i l l i n s , C h e m i s t r y , B i o l o g y , Academic P r e s s , New York, p. 27, ( 1 9 7 2 ) . 11. J . Chamberlain, J . D . Coombes, D. D e l l , J . M . Fromson, R . J . I n g s , C.M. Macdonald and J . McEwen, J . A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 6 9 , ( 1 9 8 0 ) . 1 2 . F. Esmieu, J. G u i b e r t , H . C . R o s e n k i l d e , I. Ho and A. L e Go, J . A n t i m i c r o b i a l Chemotherapy, 6 , Suppl. A, p. 83, ( 1 9 8 0 ) . 13. K. P . Fu, P . Aswapokee, I . H o , C . M a t t h i j s s e n and H . C . Neu, J . A n t i m i c r o b . Agents Chemother, 1 6 , 592,(1979).

1 4 . E. S c h r i n n e r , M. L i m b e r t , L. P e n a s s e and A. Lutz J. A n t i m i c r o b i a l Chemotherapy, 6 , Suppl. A , p. 25, (1980).

15. B.P.

(1980) "The B r i t i s h Pharmacopeia" Her M a j e s t y S t a t i o n e r y O f f i c e , Cambridge ( 1 9 8 0 ) .

FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

168

16. D.S. Reeves, L.O. White, H.A. H o l t , D . B a h a r i , M. J . Bywater and R . P . Bax, J. A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 93, ( 1 9 8 0 ) .

1 7 . T. Bergan, R. S o l b e r g , Chemotherapy ( B a s e l ) ,

27

(3),

155, (1981).

18. F. Kees, E. S t r e h l , K. S e e g e r , G . S e i d e l , P. Dominiak and H. G r o b e c k e r , Arzneim. F o r s c h . , 31 (l), 3 6 2 , ( 1 9 8 1 ) . 19. R. Wise, N . Wright and P . J . W i l l s , J . A n t i m i c r o b . Agents and Chemother., 9, 526, ( 1 9 8 1 ) . 20. F . J . Muhtadi, M.M.A. Hassan and M.M. Spectroscop. l e t t . I n P r e s s (1982).

Tawakkol,

21. D.C. Grove and W.A. R a n d a l l , Assay Methods of A n t i b i o t i c s , A n t i b i o t i c Monograph, Vol. 2. M e d i c a l E n c y c l o p e d i a , N e w York, p. 3 4 , ( 1 9 5 5 ) . 22. Y . Chabbert and H. B o u l i n g e r , Rev. F r a n c . E t . C l i n . B i o l . (2 ) 636, (1957). 23. N . Lambert-Zechovsky, C . A u f r a n t , E . Bingen, C. Blunn, M.C. Proux and H . M a t h i e u , J. A n t i m i c r o b i a l Chemotherapy, 6 , S u p p l . A, p. 235, ( 1 9 8 0 ) . 24. H. Lode, B. Kemmerich, G . G r u h l k e , G. D z w i l l o , P. Koeppe and I . Wagner, J . A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A, p. 193, ( 1 9 8 0 ) . 25. W.M. Glb'ckner, U. H o f f l e r , J. K i n d l e r , G. P e t e r s and H.G. S i e b e r t h , J . A n t i m i c r o b i a l Chemotherapy, 5, Suppl. A , p. 219, ( 1 9 8 0 ) . 26. J . P . F i l l a s t r e , A. L e r o y , G. Humbert and M. Godin, J . A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 1 0 3 , (1980). Acknowledgment The a u t h o r s would l i k e t o t h a n k R o u s s e l - U c l a f , P a r i s , France f o r kindly providing cefotaxime sample.

CEFOXITIN, SODIUM Gerald S. Brenner

I . Introduction 1.1 History 1.2 Name, Formula, Molecular Weight 1.3 Definition 1.4 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Magnetic Resonance Spectra 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Solubility 2.7 Crystal Properties 2.8 Hygroscopicity 2.9 Acid Dissociation Constant 3. Synthesis 4. Stability-Degradation Products 4.1 Bulk Stability 4.2 Solution Stability 5 . Pharmacokinetics and Metabolism 5.1 Pharmacokinetics 5.2 Metabolism 5.3 Intramuscular Absorption and Bioavailability 5.4 Effect of Probenecid 6. Methods of Analysis 6.1 Identification Tests 6.2 Ultraviolet Spectrophotometric Analysis 6.3 Chromatographic Analysis 6.4 Perchloric Acid Titration 6.5 lodometric Assay 6.6 Microbiological Assay 6.7 Hydroxylamine Assay 7. Determination in Biological Fluids 8. References

Analytical Profiles of Drug Substances Volumc I I

I69

170 170 170 170 170 171 171 171 179 179 182 182 183 183 183 183 185 185 186 187 187 187 187 188 188 188 189 189 190 190 190 190

191 192

Copyriaht Q 1982 by The American Pharmaceutical Association

ISBN &12-260(111-9

170

GERALD S. BRENNER

1.

Introduction 1.1 Historv Sodium cefoxitin, a semi-synthetic cephamycin antibiotic was synthesized, tested and developed by the Merck Sharp and Dohme Research Laboratories. Cefoxitin is active against Gram-positive and Gram-negative bacteria ( 1 ) and is therapeutically important because of its resistance to destruction by bacterial 6-lactamase (2,3,4).

1.2 Name, Formula, Molecular Weight The Chemical Abstracts name for sodium cefoxitin (MK-306) is 3-[: [Aminocarbonyl)oxylmethyl I-7-methoxy-8-0x07~~2-thienylacetyl)aminol-5-thia-l-azabicyclo~4.2.Oloct-2ene-2-carboxylic acid sodium salt. The CAS registry no. is

33564-30-6.

Other names include 3-carbamoyloxymethyl-7a-methoxy7~-(2-thienylacetamido)-3-cephem-4-carboxylic acid sodium salt, sodium 3-carbamoyloxymethyl-7-methoxy-7-[2-(2-thienyl~ acetamidol-3-cephem-4-carboxylate and sodium (6R,7R)-3-(hydroxymethyl)-7- -methoxy-8-oxo-7-[2-(2-thienyl)acetamidol-5thia-l-azabicyclo[4.2.Oloct-2-ene-2 carboxylate carbarnate (ester)

.

‘1

6H16N3Na07S2

Molecular Weight: 449.44

1.3 Definition

Sodium cefoxitin, unless specified otherwise, is defined as the crystalline, non-solvated salt form of the compound. Cefoxitin when specified refers to the free acid.

1 .4

Appearance, Color, Odor White to off-white granules or powder having a slight characteristic odor.

CEFOXITIN, SODIUM

171

Physical Properties 2.1 Infrared Spectrum The infrared spectrum of c e f o x i t i n presented i n Figure 1 was taken i n a KBr p e l l e t using a P e r k i n Elmer Model 621 infrared spectrophotometer. A list of t h e assignments made f o r some of t h e c h a r a c t e r i s t i c bands is given i n Table I ( 5 ) .

2.

Table I IR Spectral Assignments f o r Cefoxitin

Frequency (ern-')

Assignment various N-H s t r e t c h OH s t r e t c h B-lactam C=O s t r e t c h carboxyl C=O s t r e t c h carbamate C=O s t r e t c h amide C=O stretch various -CH2 bends carbamate C-0 s t r e t c h

3200-3500 3000 (broad) 1780 1715 1680 1660 1420 1080

The infrared spectrum of sodium c e f o x i t i n taken a s a mineral o i l m u l l is shown i n Figure 2. Magnetic Resonance Spectra 2.21 Proton Spectrum The proton magnetic resonance spectrum of c e f o x i t i n presented i n Figure 3 was obtained using a Varian A-60A spectrometer with DMSO-d6 a s t h e solvent. The s p e c t r a l assignments a r e given i n Table I1 ( 6 ) . The proton magnetic resonance spectrum f o r sodium c e f o x i t i n i n DMSO-d6 is shown i n Figure 4 . 2.2

Table I1 Proton Magnetic Resonance Assignments f o r Cefoxitin ppm ( 6 ) 9.38

Relative I n t e n s i t y 1

Multiplicity Singlet

Assignment 0

II

7.34

G -85-7.05

1 2

Mu 1ti p l et Mu1t i p l e t

-C-NHthienyl-a thienyl-B

0 g

c

I 0

o O/o

I 0 W

c

1 0

t

I 0 N

0

0 'z -O 0 9 -

a0

0 -0

-%

0

3 3N V l1I W S NV t l l

172

a,

u

c

.rl

x

U

C

V

ir

m

!-I H

w C

I

r 0

P-

0 0

O 0 al

H 8 0

0 0

2 0 0

:: 0 0 0

w

I73

174

A 1m

FIGURE 4

I I

I

.

J .

8.0 Figure 4 .

I

.

7.0

I

.

,

.

,

.

,

,

I

.

6.0

A .

.

.

I

,

5.0

,

.

,

,

.

.

I

,

.

4.0 PPM

,

.

,.

,

1

,

,

3.0

.

,

,

.

.

1

.

2.0

, .

,

.

.

.

1

,

.

10

.

.

.

I

0

Proton Magnetic Resonance Spectrum of Sodium Cefoxitfn in DMSO-d 6 '

176

GERALD S. BRENNER

Table I1 (cont'd) ppm

(6

Relative Intensity

1

6.52

2

Multiplicity

Assignment

Singlet

0

I1

5.13 4.49-5.0

1 2

0-c-NH2 6-H

Singlet Mu1tiplet

0

II

-CH20C0

}

3.08-3.80 3'83 3.40

6-7

Singlet Mu1tiplet Singlet

II

thien yl-CH2-C

b-CH

7-OC~;~

2.22 Carbon-13 Spectrum The carbon-13 magnetic resonance spectra of cefoxitin presented in Figures 5 and 6 were obtained using a Varian CFT-20 spectrometer with DMSO-d as the solvent at M. The spectra assignments are a concentration of 0.5 given in Table I11 (7).

P

Table I11 Carbon-13 Magnetic Resonance Assignments for Cefoxitin

ppm( 6 )

170.4 162.5 160.3 156.3 136.5 126.5 126.4}

Assignment amide C=O carboxyl C=O C8, lactam C=O carbamate C=O c21 C31 and C4l

c a r boxy I

\

onide

C

\

c corbomok

7

J1

ow3

I

I c,

175

I60

Figure 6.

PPM

I25

C-13 WiA Spectrum of Cefoxitin in DMSO-d

6'

CEFOXITIN, SODIUM

179

Table I11 (cont'd) Assignment

ppm ( 6 ) 125.8 125.5 124.9 95.1 62.8 61.9 52.5

c j1 c7 c6 C OEB, -I

0

II

35.9

thien yl-CH2-C-

25.7

c4

U l t r a v i o l e t Spectrum The u l t r a v i o l e t absorDtion sDectrum o f c e f o x i t i n i n pH 7.0 phosphate buffer is chabacterized by a peak a t about 2 5 nm and a shoulder a t about 262 nm (Figure 7) with A?$m values of approximately 330 and 209 respect i v e l y . The shoulder a t 262 nm is d u e t o t h e 3-cephem chromophore and t h e maximum a t 235 nm is due to thienyla c e t y l s i d e chain. 2.3

2.4

Mass Spectrum

The mass spectrum o f c e f o x i t i n was obtained u s i n g a Finnigan 3200 mass spectrometer i n t h e e l e c t r o n impact moie w i t h an ionizing energy of 70 eV and a probe temperature of 175OC. The output from t h e mass spectrometer was analyzed (8) and presented i n t h e form of a bar graph shown i n Figure

8. The ion of highest mass seen i n t h e spectrum o f c e f o x i t i n f r e e acid (molecular weight 427) is m/e 366, which corresponds t o t h e lactone formed from t h e

0

~cr''

C H 2-CO N H OCH3

0

N N

0 Cefoxit i n 1ac tone

CH2

d

\

\

\

\

I

250 nm

Figure 7.

300 nm

UV Spectrum of Cefoxitin in pH 7 Phosphate Buffer. Concentration: 2.75 mg/100 ml. 180

c 0,

D

Q d

AlISN31NI 3 A l l V l 3 Y

181

“1 D

Ln (3

D D (3

D u)

N

D D

N

D

2

0

+

0

D In

c

.d U

0

x

*r(

CJ

a

u 0

w

c

0

3

U

.rl

rn

0

4

a

d

GERALD S. BRENNER

182

carboxylic acid and carbamate group. The n e x t ion (m/e 322) corresponds t o l o s s o f C02 from the l a c t o n e . Scission o f t h e s i d e chain amide bond of t h e l a c t o n e g i v e s m/e 241, to give m/e 210. A t lower mass one s e e s which l o s e s CH20 J

m /e 124,

m/e

@CH=C=O

+

112,

m/e 97,

2.5

Optical Rotation The s p e c i f i c r o t a t i o n o f a 1% (w/v) sodium c e f o x i t i n s o l u t i o n i n methanol determined a t 509 nm and 25OC i s +210° + 4' calculated on an anhydrous and solvent f r e e b a s i s .

2.6

Solubility The s o l u b i l i t y o f sodium c e f o x i t i n i n t h e following s o l v e n t s a t room temperature is s t a t e d i n terms of t h e U.S.P. d e f i n i t i o n s : v e r y s o l u b l e i n water, s p a r i n g l y soluble i n methanol and dimethylformamide, s l i g h t l y s o l u b l e i n ethanol and i n s o l u b l e i n e t h e r , chloroform, acetone aromatic and a l i p h a t i c hydrocarbons. The s o l u b i l i t y of c e f o x i t i n an organic acid with l i m i t e d aqueous increases with increasing pH. A t pH s o l u b i l i t y is about 0.3 mg/ml and a t value is about 25 mg/ml ( 9 ) .

i n water is t y p i c a l of s o l u b i l i t y and 1 , t h e measured pH 4 , t h e observed

CEFOXITIN, SODIUM

183

2.7

Crvstal ProDerties 2.571 C r y s t a l l i n i t y Sodium c e f o x i t i n exists i n s e v e r a l solvated and i n a desolvated c r y s t a l l i n e form. An amorphous form has a l s o been i d e n t i f i e d . X-ray powder d i f f r a c t i o n p a t t e r n s and polarized l i g h t microscopy can d i s t i n g u i s h c r y s t a l l i n e from amorphous forms ( 1 0 ) . 2.72

X-Ray Powder Diffraction The x-ray powder d i f f r a c t i o n p a t t e r n o f c r y s t a l l i n e desolvated sodium c e f o x i t i n was obtained with a Philips-Norelco d i f f r a c t o m e t e r , using copper Kcc r a d i a t i o n . Samples of t h i s form show broad d i f f r a c t i o n l i n e s suggesting t h a t t h e s o l i d is m i c r o c r y s t a l l i n e and composed of extremely small c r y s t a l l i t e s ( 1 1 ) . The amorphous form shows no d i s t i n c t X-ray powder d i f f r a c t i o n p a t t e r n . 2.73

D i f f e r e n t i a l Scanning Calorimetry C r v s t a l l i n e sodium c e f o x i t i n e x h i b i t s a decomposition exotherm a t approximately 19O0-2OO0C by d i f f e r e n t i a l scanning calorimetry (DSC). The amorphous form does not e x h i b i t any well-defined thermal t r a n s i t i o n s by DSC below 35OoC. Gradual decomposition of t h i s s o l i d is observed by DSC a s temperatures increase over 15OoC ( 1 1 ) . 2.8

Hygroscopicity The water uptake o f sodium c e f o x i t i n a s a function of r e l a t i v e humidity was s t u d i e d a t ambient temperature ( 1 1 ) . Equilibrium water levels were approximately I-2% a t 35% RH, 4-6% a t 47% RH and 15-17% a t 76% RH. 2.9

Acid Dissociation Constant The d i s s o c i a t i o n constant o f c e f o x i t i n derived from aqueous t i t r a t i o n (12) and s o l u b i l i t y d a t a (9) a r e i n good agreement and i n d i c a t e t h a t c e f o x i t i n i s a r e l a t i v e l y s t r o n g organic acid with a pKa of approximately 2.2.

3.

Synthesis Cefoxitin has been prepared by chemical modification of cephamycin C , a n a t u r a l l y occuring a n t i b i o t i c produced by Streptomyces lactamdurans (13, 1 4 ) . This r o u t e i s presented i n Figure 9. Cepharnycin C (I) is tosylated t o t h e N-tosyl d e r i v a t i v e (11) and then e s t e r i f i e d w i t h methyl chloromethyl ether t o y i e l d t h e methoxymethyl ester (111). The ester i s t h e n t r e a t e d with 2-thienylacetyl c h l o r i d e t o exchange t h e aminoadipoyl s i d e chain f o r t h i e n y l a c e t y l . The ester protecting group i s then removed w i t h acid t o y i e l d c e f o x i t i n (IV).

r

I

1

II: CICH20C H3

Figure 9 S y n t h e s i s of C e f o x i t i n from Cephamycin C

CEFOXITIN, SODIUM

185

C e f o x i t i n h a s a l s o been p r e p a r e d b y t o t a l s y n t h e s i s (15) and v i a t h e a c y l a t i o n and m e t h o x y l a t i o n of 7-amino c e p h a l o s p o r a n i c a c i d ( 1 6 , 17, 18, 19). 4.

Stability-Degradation Products 4.1 Bulk S t a b i l i t v A t room t e m p e k a t u r e sodium c e f o x i t i n i s s t a b l e for a t l e a s t t h r e e y e a r s when p r o t e c t e d from m o i s t u r e . A t elev a t e d t e m p e r a t u r e , t h e s o l i d e x h i b i t s a b i p h a s i c decomposit i o n p a t t e r n t y p i f i e d by an i n i t i a l more r a p i d d e c o m p o s i t i o n p e r i o d f o l l o w e d by a slower d e c a y p e r i o d ( 2 0 ) . T h i s phenomenon may b e related t o d e g r a d a t i o n of c e f o x i t i n b y low l e v e l s of w a t e r i n t h e sample. S i n c e 6 - l a c t a m c l e a v a g e is waterconsuming, t h e e x t e n t of t h i s d e g r a d a t i o n pathway is l i m i t e d by a v a i l a b l e water i n t h e s o l i d . Amorphous sodium c e f o x i t i n h a s been shown t o be c o n s i d e r a b l y less s t a b l e t h a n i t s c o r r e s p o n d i n g c r y s t a l l i n e form ( 2 0 ) . A t e m p e r a t u r e d e p e n d e n t d i s c o l o r a t i o n of t h e s o l i d h a s been n o t e d . The d i s c o l o r a t i o n i s n e g l i b l e a t 5OC and becomes greater a t e l e v a t e d t e m p e r a t u r e . I t h a s been shown t h a t an i n e r t a t m o s p h e r e ( a r g o n or n i t r o g e n ) m a r k e d l y d e c r e a s e s t h i s change. T h i s development of color is n o t d i r e c t l y r e l a t e d t o l o s s of p o t e n c y , i . e . c o n s i d e r a b l e d i s c o l o r a t i o n c a n o c c u r w i t h no m e a s u r a b l e loss of p o t e n c y . S e v e r a l d e g r a d a t i o n p r o d u c t s of s o l i d sodium c e f o x i t i n h a v e been i d e n t i f i e d . From material s t o r e d a t 6OoC for t h r e e d a y s , two compounds h a v e been i s o l a t e d and identified.

I,p

186

GERALD S. BRENNER

Solution S t a b i l i t y The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n has been studied i n aqueous b u f f e r s i n t h e pH range 3 t o 9 ( 2 0 ) . The degradation of sodium c e f o x i t i n i n t h i s pH range follows apparent f i r s t - o r d e r k i n e t i c s . Maximum s t a b i l i t y i n water i s i n t h e pH range o f 5-7. Under these pH c o n d i t i o n s , sodium c e f o x i t i n undergoes 10% chemical l o s s i n two days a t 25OC. Ten percent l o s s a t pH 3 occurs i n about 40 hours and a t pH 9 i n about 14 hours. TLC s t u d i e s were c a r r i e d o u t during k i n e t i c r u n s . P a t t e r n s become complex suggesting t h a t t h e i n i t i a l B-lactam hydrolysis product i s unstable and s u s c e p t i b l e t o transformation t o a considerable number of secondary products (20). 4.2

The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n was a l s o studied a f t e r c o n s t i t u t i o n with frequently used I . V . infusions and admixture w i t h comnonly used I . V . and I.M. a d d i t i v e s (21 1. S t a b i l i t y i n t h e s e systems was e s s e n t i a l l y t h e same a s t h a t observed f o r unbuffered s o l u t i o n s . I n t h e s e s t u d i e s , sodium c e f o x i t i n was shown t o maintain potency i n s o l u t i o n f o r a t l e a s t 30 days a t 5OC and f o r 30 weeks when stored i n t h e frozen s t a t e . I n an attempt t o i s o l a t e degradation products formed i n s o l u t i o n , a 10% aqueous s o l u t i o n of sodium c e f o x i t i n was heated f o r four days a t 8OoC and then subjected t o preparative TLC. F r a c t i o n s i s o l a t e d were examined by NMR, mass spectrometry and i n f r a r e d . The following compounds were i d e n t i f i e d .

Thiophene-2-acetic

acid

Thiophene -2-

acetamidr

CON+

N- ( 2'-mothoxyacotamido) thiophene2- ace t am ido

CEFOXITIN, SODIUM

187

5.

Pharmacokinetics and Metabolism The pharmacokinetics and metabolism of sodium c e f o x i t i n i n humans following parenteral administration have been t h e subject of a number of i n v e s t i g a t i o n s and reviews (22,31,32).

5.1

Pharmacokinetics Followina intravenous administration (bolus o r i n f u s i o n ) , c e f o x i t i n i s d i s t r i b u t e d r a p i d l y between serum and t i s s u e and e x h i b i t s a terminal serum h a l f - l i f e of 30 t o 50 m i n u t e s . Total body clearance of c e f o x i t i n ranges from approximately 250 m l t o 350 m l / m i n while r e n a l clearance i s approximately 200 t o 300 m l / m i n . Urine contains a t l e a s t 90% of t h e dose a s unchanged drug and less than 5% of t h e dose i s eliminated by metabolism and b i l i a r y clearance (23,24,25). The d i s p o s i t i o n k i n e t i c s are f i r s t o r d e r , showing no e f f e c t of dose (0.25 t o 3 g.) or infusion r a t e . Multiple dose regimens i n t h i s range given every f o u r hours do not cause accumulation i n healthy volunteers. The volume of d i s t r i b u t i o n i n t h e vascular compartment is about 8 l i t e r s (26-34). These d a t a a r e adequately described by a two-compartment open model w i t h elimination occurring from t h e c e n t r a l compartment. 5.2

Metabolism Sodium c e f o x i t i n i s not metabolized appreciably i n man. Urine samples from s e v e r a l human s t u d i e s were separated by HPLC and TLC techniques. These s t u d i e s show t h a t more than 90% of t h e c e f o x i t i n administered by e i t h e r t h e intravenous o r intramuscular route i s recovered i n t h e u r i n e a s i n t a c t drug. A microbiologically i n a c t i v e metabolite, descarbamoyl c e f o x i t i n , was found t o t h e extent of 1-6% i n some i n d i v i d u a l s , 2 t o 4 hours post dosing (26,271. This metabolite was not found i n t h e u r i n e o f a l l s u b j e c t s .

Descarbarnoyl c e f o x i t i n

GERALD S . BRENNER

188

5.3

Intramuscular Absorption and B i o a v a i l a b i l i t y Sodium c e f o x i t i n is raDidly and completely absorbed following intramuscular administration. Peak serum levels a r e a t t a i n e d i n 30 m i n u t e s o r less and 85 t o 95% of t h e i . m . dose is recovered i n u r i n e within 12 hours of administration. Intramuscular administration o f sodium c e f o x i t i n with either 0.5 o r 1.0% l i d o c a i n e hydrochloride a s a d i l u e n t has no apparent e f f e c t on t h e b i o a v a i l a b i l i t y o f t h e an tibiotic (32,35>.

5.4

E f f e c t o f Probenecid The concurrent o r a l o r intravenous administration of probenecid with i . m . o r i . v . i n j e c t i o n s of c e f o x i t i n has a l a r g e influence on t h e time course o f t h e a n t i b i o t i c i n serum (26,36). Probenecid administered intravenously concurrent with c e f o x i t i n i n c r e a s e s t h e serum h a l f - l i f e o f c e f o x i t i n from approximately 40 minutes t o 80 m i n u t e s and reduces t h e r e n a l clearance of t h e drug from 200-300 ml/min t o less than 100 m l / m i n . 6.

Methods o f Analysis 6 . 1 I d e n t i f i c a t i o n Tests U l t r a v i o l e t spectrophotometry i s used t o i d e n t i f y sodium c e f o x i t i n . The spectrum o f a sample dissolved i n pH 6.0 phosphate buffer scanned from 220 t o 310 nm compares q u a l i t a t i v e l y t o t h a t of a c e f o x i t i n standard s i m i l a r l y tested. The i d e n t i t y o f sodium c e f o x i t i n is a l s o established by i n f r a r e d spectroscopy. The i n f r a r e d absorbance spectrum of a s o l i d sample prepared e i t h e r a s a potassium bromide d i s c o r mineral o i l m u l l is compared t o a standard sample prepared i n an i d e n t i c a l manner. A color test has been employed f o r t h e d e t e c t i o n of c e f o x i t i n i n s o l u t i o n (37). To t h e r e s i d u e obtained by drying a s o l u t i o n containing 10-50 mcg of c e f o x i t i n i s added 1-2 m l of 0.01% ninhydrin i n concentrated sulfuric acid and t h e color is allowed t o develop a t room temperature. A vivid blue c o l o r appears i n a few m i n u t e s . Other cephalosporin a n t i b i o t i c s do not give t h e same c o l o r response.

CEFOXITIN, SODIUM

189

Cefoxitin and f i f t e e n other cephalosporins have been i d e n t i f i e d by thin-layer chromatography coupled with color r e a c t i o n s (38) and by spectroscopic methods (39). 6.2

U l t r a v i o l e t Spectrophotometric Analysis I n t a c t sodium c e f o x i t i n e x h i b i t s a UV absorption band near 262 nm a t t r i b u t e d t o t h e OX-N-C=C linkage i n t h e molecule. Beta-lactam r i n g opening l e a d s t o disappearance of t h i s absorption band. This observation is t h e b a s i s of a q u a n t i t a t i v e assay f o r c e f o x i t i n which i s s t a b i l i t y indicating. Calculation of i n t a c t compound i s based on t h e net absorbance a t 262 nm of t h e sample and of t h e standard i n pH 6.0 phosphate buffer a s determined by s u b t r a c t i n g a base l i n e correction a t 262 nm from t h e maximum a t t h e same wavelength. The correction i s found by extending t o 262 nm t h e s t r a i g h t portion of t h e UV curve between 340 and 300 nm. 6.3

Chromatographic Analysis 6.31 Thin Layer Chromatography Thin layer chromatography using t h e system chloroform/acetone/formic acid (10:9: 1 ) w i t h 0.25 rnm s i l i c a gel p l a t e s has been employed f o r both sodium c e f o x i t i n and the f r e e acid. The a i r dried p l a t e is sprayed w i t h 0.2% pdimethylaminocinnamaldehyde i n methanol/conc. s u l f u r i c acid ( 4 : l ) and heated a t 105' f o r f i v e m i n u t e s . The Rf f o r c e f o x i t i n i n t h i s sytem is approximately 0.45. Cefoxitin has a l s o been chromatographed on s i l i c a w i t h developing s o l v e n t s of n-butanol/water/acetic acid ( 4 : l : l ) and benzene/methanol/acetic acid (50:10:6) giving approximate Rf values of 0.7 and 0.2 r e s p e c t i v e l y . Detection can be accomplished by fluorescence quenching or iodine s t a i n i n g . 6.32

High Performance Liquid Chromatography Several HPLC procedures have been developed t o s e p a r a t e c e f o x i t i n from process i m p u r i t i e s and degradates. A system frequently used is described below. Mobile Phase: containing 1% a c e t i c acid.

20% a c e t o n i t r i l e i n water

Column : Ten micron, microporous oct,adecylsilane bonded reversed phase packing i n a 3.9 mn X 30 cm column. Column temperature and pressure a t 25OC ( o r ambient) and 1000-1500 p s i g , respectively. Flow r a t e of I .O-l.3 ml/min. Detection:

U.V.

a t 254 nm

GERALD S. BRENNER

190

Procedure: Aliquots (10 mcl) o f a sample s o l u t i o n containing 0.25 m g h l i n 0.02 M pH 7 phosphate f o r c e f o x i t i n is buffer a r e i n j e c t e d . The r e t e n t i o n approximately 10-15 m i n u t e s .

time

6.4

Perchloric Acid T i t r a t i o n Sodium c e f o x i t i n can be determined by non-aqueous t i t r a t i o n . An accurately weighed sample of about 800 mg i s dissolved i n about 100 m l of g l a c i a l a c e t i c acid and t i t r a t e d potentiometrically w i t h standardized 0.1N p e r c h l o r i c acid i n s p e c t r a l grade dioxane. An e l e c t r o d z p a i r i s employed c o n s i s t i n g of a calomel e l e c t r o d e which has been r e f i l l e d w i t h 0.1N LiC104 i n a c e t i c anhydride a s t h e i n d i c a t i n g e l e c t r o d e , and a platinum r i n g a s t h e r e f e r e n c e electrode. Iodometric Assay Sodium c e f o x i t i n bulk chemical and formulations can be determined by iodometric assay. I n t h e assay, a l i q u o t s of t h e sample and o f a s u i t a b l e c e f o x i t i n standard s o l u t i o n a r e hydrolyzed f o r 20 minutes with 1 N NaOH and then a c i d i f i e d t o pH 3.0 with a c e t a t e bufTer and I N H C 1 . Then, 0.01N iodine s o l u t i o n is added and, a f t e r a 2 h i n u t e w a i t , t h e excess iodine is t i t r a t e d with 0.01N sodium t h i o s u l f a t e s o l u t i o n t o a s t a r c h end p o i n t . A blank determination i s made a t t h e same time on a l i q u o t s o f both sample and standard s o l u t i o n s t r e a t e d only with buffer and 0.01N iodine and allowed t o stand f o r 20 minutes. 6.5

An automated iodometric assay has been developed based on the manual method described above w i t h t h e exception t h a t excess iodine is measured c o l o r i m e t r i c a l l y r a t h e r than by t h i o s u l f a t e t i t r a t i o n ( 4 0 ) . 6.6

Microbiological Assay For bulk and formulated products an agar d i f f u s i o n ( p l a t e ) assay can be conveniently c a r r i e d out using Staphylococcus aureua a s t h e t e s t organism ( 4 1 )

.

6.7

Hydroxylamine Assay Sodium c e f o x i t i n can be determined by means of t h e colored complex formed between f e r r i c ion and t h e hydroxamic acid formed by t h e a c t i o n o f hydroxylamine on t h e betalactam ( 4 2 ) . This method has been s u c c e s s f u l l y automated

CEFOXITIN, SODIUM

191

and has been specified by t h e FDA a s t h e d e f i n i t i v e assay method f o r c e r t i f i c a t i o n of t h e a n t i b i o t i c .

7.

Determination i n Biological F l u i d s High performance l i q u i d chromatography has been u t i l i z e d f o r t h e determination of c e f o x i t i n i n b i o l o g i c a l f l u i d s . Cefoxitin and i t s descarbamoyl metabolite have been q u a n t i t a t i v e l y analyzed i n human urine employing an anionMore r e c e n t l y exchange column w i t h U.V. detection ( 2 3 ) . Wheeler, e t a l . (43) have employed HPLC u t i l i z i n g a C-18 reversed phase packing and a solvent system of a c e t o n i t r i l e / a c e t i c acid/O. 005M potassium dihydrogen phosphate (25/0.5/74.5, v / v / v ) f o r the q u a n t i t a t i o n of c e f o x i t i n i n serum and s a l i v a . An HPLC method has been developed f o r t h e determination of c e f o x i t i n i n serum which l e n d s i t s e l f t o automation ( 4 4 ) . The system is described below: Mobile Phase: 11% methanol i n pH 6.86 buffer containing 1% a c e t o n i t r i l e . Column: Ten micron C-8 reversed phase packed i n a 3.9 mn x 25 cm column with a disposable precolumn i n l i n e and a precolumn i n the i n j e c t i o n loop. m l / m i n is used. Detection:

U.V.

A flow r a t e o f 3.0

a t 254 nm.

Procedure: Serum samples (100 1.111 a r e t r e a t e d with 100 1 of i n t e r n a l standard (aqueous cefmetazole, 15 u l / m l and 75 u 1 10% t r i c h l o r o a c e t i c a c i d . SarnDles a r e m i x e d , allowed t o stand for 15 m i n u t e s and centkifuged t o remove p r e c i p i t a t e d protein. For automated a n a l y s i s , c l e a r supernatant is t r a n s f e r r e d t o microcentrifuge tubes. A standard curve i s generated by spiking blank serum with appropriate levels of c e f o x i t i n and then processing a s described above. Cefoxitin i n b i o l o g i c a l f l u i d s has a l s o been determined microbiologically by t h e cup-plate diffusion-technique using e i t h e r Staphylococcus aureus MB2876 (26,27) o r B a c i l l u s s u b t i l i s MB36 (35) a s t h e t e s t organism.

GERALD S.BRENNER

192

8.

References

1 . H. R . Onishi, D. R. Daoust, S. B. Zimnerman, D. Hendlin and E. 0. Stapley, "Abstracts, XI1 I n t e r s c i e n c e Conference on Antimicrobial Agents and Chemotherapy", A t l a n t i c C i t y , N.J., 1972, p. 77 and preceding two a b s t r a c t s . 2. G. Darland and J . Birnbaum, Antimicrob. Agents Chemother. 1 1 , 725 (1977).

3. H. R . Onishi, D. R . Daoust, S. B. Zimerman, D. Hendlin 5 , 38 and E. 0. Stapley, Antirnicrob. Agents Chemother., (1974).

4 . H. C. Neu, Antimicrob. Agents Chemother.,

6,

170 (1974).

5. J . A. Ryan and J . Stevenson, Merck Sharp and Dohme Research Laboratories, personal communication. 6. A. Douglas, Merck Sharp and Dohme Research Laboratories, personal comnunication.

7. A. Douglas, Merck Sharp and Dohme Research Laboratories, personal communication.

8. W. J . A. Vandenheuvel, Merck Sharp and Dohme Research Laboratories, personal communication.

9. J . A. McCauley and A. Shah, Merck Sharp and Dohme Research Laboratories, personal communication. 10. E. R. Oberholtzer and B. Singleton, Merck Sharp and Dohme Research Laboratories, personal communication.

1 1 . E. R. Oberholtzer, Merck Sharp and Dohme Research Laborat o r i e s , personal communication.

12. G . Bicker, Merck Sharp and Dohme Research Laboratories, personal communication.

13. S. Karady, S. H. P i n e s , L. M. Weinstock. F. E. Roberts, G. S. Brenner, A. M. Hoinowski, T. Y. Cheng and M. S l e t z i n g e r , J . Am. Chem. SOC. 94, 1410 (1972). 14. L. M. Weinstock (Merck & Co., I n c . ) Fr 2,253,022; Ger Offen 2,456,528; Jap. K75,105,687, Neth 7,414,820

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193

15. R. W. R a t c l i f f e and B. G. Christensen, Tetrahedron Lett 4 6 , 4645 (1973); 46, 4649(1973); 46, 4653 (1973). 16. G. G. Hazen (Merck and Co., Inc.) US 3,780,033.

17. B. C. Christensen and L. D. Cama (Merck & Co., Inc.) Fr 2,163,144; Ger Offen 2,258,278. 18. B. C. Christensen and R. A. Firestone (Merck & Co., I n c . ) US 3,775,410. 19. B. G . C h r i s t e n s e n e t . a].. (Merck & Co., Inc.) Brit 1,348,984; Ger Offen 2,143,331. 20. E. R . Oberholtzer and G . S. Brenner, J. Pharm. S c i . , 68, 863 ( 1979). 21. M. J . O'Brien, J . B. Portnoff, and E. M. Cohen, Am. J . Hosp. Pharm 36, 33 (1979). 22. K . C. Kwan and J . D. Rogers i n 6-lactam A n t i b i o t i c s (Handbook o f Experimental Pharmacology) ed. A. L. Demain. Springer-Verlag, Heidelberg, i n press.

23. R. P. Buhs, T. E. Maxim, N. Allen, T. A. Jacob and F. J . Wolf, J . Chromatog 99, 609 (1974). 24. A. M. Geddes, L. P. Schnurr, A. P. B a l l , D. McChie, G . R. Brookes, R . Wise and J . Andrews, Br. Med. J . 1 , 1126 ( 1977).

25. M. N . Logan, R . Wise and R. P. Grimley, J . Antimicrob. Chemother. 5 , 620 (1979). 26. C. S. Goodwin, E. B. Raffery, A. D. Goldberg, H. Skeggs, A. E. T i l l , and C. M. Martin, Antimicrob. Agents 6, 338 (1979). Chemother. -

27. P. F. Sonneville, R. R . Kartodirdjo, H. Skeggs, A. E. T i l l and C. M. Martin, Europ. J . C l i n . Pharmacol. 9, 397 (1976). 28, 28. C. Simon, E. Meyer and V. Malerizyk, Arm, Forsch. 1541 (1978).

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29. G . J . Pazin, S. N. Schwartz, M. Ho, J . A. Lyon and A. W. Pasculle, Rev. I n f e c t . D.S. 1 . 189 (1979). 30. J.J. Schrogie, R . 0. Davies, K . C. Yeh, D. Rogers, G . I . Holmes, H. Skeggs and C. M. Martin, J . Antimicrob. 4 , :Suppl. B, 69 (1978). Chemother. -

31. J . J . Schrogie, J . D. Rogers, K . C. Yeh, R. 0. Davies, C. I . Holmes, H. Skeggs and C. M. Martin, Rev. I n f e c t . Disc. 1 , 90 (1979).

32. W. Brumfitt, J . Kosmidis, J . M. T. Hamilton-Miller and J . N. G . G i l c h r i s t , Antimicrob. Agents Chemother. 6 , 290 (1974).

33. A. M. Brisson, J.B. F o u r t i l l a n , D. Barthes, P. Courtois and B. Bezq-Giraudon, Therapie

35,

209 (1980).

34. R . Wise, B. Cadge, A. P. Gillett and A. Bhamjee, J . Infection 1 : Suppl. 1 , 49 (1979).

35. P. F. Sonneville, K . S. Albert, H. Skeggs, H. Centner, K . C. Kwan and C. M. Martin, Europ. J . C l i n . Pharmacol.

12, 273 (1977). 36. P. H. Vlasses, A. M. Holbrook, J . J . Schrogie, J . D. Rogers, R. K . Ferguson and W. B. Abrams, Antimicrob Agents Chemo. Ther. 1, 847 (1980).

37. J . R. Carlin and T. A. Jacob, Merck Sharp and D o h e Research Laboratories, personal comnunications. 40. J . M. Konieczny, Merck Sharp and Dohme Research Laborat o r i e s , personal communications. 41. Code of Federal Regulations, Sections 4 4 2 . 1 4 a ( b ) ( l ) ( i ) and 436.105. 42. Code o f Federal Regulations, Sections 4 4 2 , 1 4 a ( b ) ( l ) ( i i ) and 4 4 2 . 4 0 ( b ) ( l ) ( i i ) .

43. L. A. Wheeler, M. de Meo, B. D. Kirby, R. S. Jerauld and S. M. Finegold, J . Chromatog. 183, 357 ( 1980). 44. R. S h a f f e r , Merck Sharp and Dohme Research Laboratories, personal comnunication. L i t e r a t u r e Reviewed t o June 1981

CEFOXITIN, SODIUM

Acknowledgement The author wishes t o thank Mrs. E. Moyer f o r typing t h e manuscript, Ms. F. R. Berg and Ms. A. M. Hendrick f o r l i t e r a t u r e r e t r i e v a l work and Mr. C. Dillman and Ms. N . G i l b e r t f o r preparation of the f i g u r e s .

195

CLOFIBRATE Mahmoud M . A. Hassan and Aida A. Elaxxouny

1.

2.

3. 4. 5. 6.

7. 8.

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Appearance, Color, Taste, Odor Physical Properties 2.1 Boiling Range 2.2 Density 2.3 Refractive Index 2.4 Solubility 2.5 Spectral Properties Synthesis Metabolism Pharmacology Methods of Analysis 6.1 Elemental Analysis 6.2 Identification Tests 6.3 Purity Tests 6.4 Official Methods 6.5 Ultraviolet Spectrophotometry 6.6 Thin Layer Chromatography 6.7 Thin Layer-Gas Liquid Chromatography 6.8 Gas Liquid Chromatography 6.9 Gas Chromatography-Mass Spectrometry 6.10 High-Performance Liquid Chromatography Proton Magnetic Resonance Spectrometry References

Analytical Profiles of Drug Substances

Volume I I

197

198 198 198 199 199 199 199 199 199 199 199 207

209 209 21 1 21 1 21 1 21 1 212 213 214 214 214 217 218 219 22 1

Copyrilht 0 1982 by The American Phlmuceuliul h o c u t i o n

ISBN & 1 2 - ~ l l - 9

198

1.

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY Description

1.1

Nomenclature 1.11

1.12

Chemical Names a.

2-(4-chlorophenoxy) -2-methylpropanoic e t h y l ester.

b.

E t h y l 2-p-chlorophenoxy

c.

E t h y l 2-(p-chlorophenoxy)-2-methyl propionate.

d.

Propanoic a c i d , 2-(4-chlorophenoxy)-2methyl-, e t h y l ester.

acid

isobutyrate

Generic Name Clof i b r a t e

1.13

Trade Names Amotril ; Anparton ; Ateculon ; Atheropront ; A t e r i o s a n ; Atromidin ; Atromid-s ; C l a r i p e x ; Clobren-SF ; CPIB ; H y c l o r a t e ; Lipavion ; Neo-Atromid ; Normet ; R e c o l i p ; Regelan ; S e r o t i n e x ; Skleromexe ( 1 ) .

1.2

Formulae 1.21

Empirical '1 2H15'3'

1.22

Structural

CLOFIBRATE 1.23

199

CAS R e g i s t r y No. (637-07-0)

1.24

Wiswesser L i n e N o t a t i o n

(2)

GR DOXVO 2

1.3

M o l e c u l a r Weight 242.71

1.4

Appearance, C o l o r , Taste, Odor S t a b l e , c o l o r l e s s t o pale-yellow l i q u i d w i t h a f a i n t , c h a r a c t e r i s t i c o d o r and a c h a r a c t e r i s t i c t a s t e .

2.

Physical Properties 2.1

B o i l i n g Range 158-1 60 148-150

2.2

20

Density d25 : 1.138

2.3

R e f r a c t i v e Index n 2o D

2.4

- 1.144

1.500

-

1.505

Solubility Insoluble i n water, s o l u b l e i n acetone, a l c o h o l , benzene, and c h l o r o f o r m .

2.5

Spectral Properties 2.51

I n f r a r e d Spectrum The I n f r a r e d Spectrum of C l o f i b r a t e i s r e c o r d ed a s a f i l m on a Unicam Sp-1025 S p e c t r o p h o t o meter and i s shown i n F i g . 1 . The a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands i n t h e I n f r a r e d Spectrum a r e l i s t e d i n T a b l e 1.

Fig.

1.

I R Spectrum of Clofibrate as F i l m .

CLOFIBRATE

20 1

Table I I R C h a r a c t e r i s t i c s of Clof i b r a t e

-1 Frequency Cm

Assignments (3)

1750

C = 0 (ester).

1605 1590 1500

C = C (aromatic).

c-0-c

1150 1100

(C-0 s t r e t c h i n g ) .

1390 1375 (Symmetrical deformat i o n )

.

830

a r o m a t i c parad i s u b s it u t i o n

770 68 0

c-c1 Other bands c h a r a c t e r i s t i c of C l o f i b r a t e are 3020, 2970, 1290, 1250, 1190, 1030, 1020, 980 and 920 Cm-l. 2.52

U l t r a v i o l e t Spectrum (UV) The UV spectrum of c l o f i b r a t e i n methanol w a s scanned from 400-200 nm u s i n g V a r i a n Cary 219 Spectrophotometer and i s shown i n F i g . 2 . The spectrum e x h i b i t s t h r e e maxima a t 288 (708), 280 (10233) and 226 (8511).

2.53

Nuclear Magnetic Resonance Spectrum 2.531 Proton Spectrum The p r o t o n NMR S p e c t r a of C l o f i b r a t e i n d e u t e r a t e d chloroform and i n Acetone-&, were recorded on a Varian T-60 A, 60 MHz NMR S p e c t r o m e t e r , u s i n g t e t r a m e t h y l s i l a n e

202

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

as an i n t e r n a l reference.

The PMR spectrum i n d e u t e r a t e d chloroform i s shown i n F i g . 3. The PMR s p e c t r a l assignment of c l o f i b r a t e a r e g i v e n i n T a b l e 2 ( 4 ) . Table 2 PMR C h a r a c t e r i s t i c s of C l o f i b r a t e

Protons

Chemical S h i f t s C0Cl3

Acet one-D

1.20

1.23

t

1.56

1.57

s

-CH CH

4.18

4.20

q

Four a r o m a t i c pro t o n s

6.96

7.03

g

-CH2CH3

-C-(CH

)

3 2

-

s = singlet;

t

-

triplet;

6

q = quartet

2.532 S N M R Spectrum 13C NMR Spectrum of c l o f i b r a t e i n deut e r a t e d chloroform u s i n g t e t r a m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n c e w a s r e c o r ded on a J e o l FX 100, 100 MHz i n s t r u m e n t a t ambient t e m p e r a t u r e and u s i n g 10 mm sample t u b e . The d a t a c o n s i s t of 8192 d a t a p o i n t s over a 5000 Hz S p e c t r a l Width. The c o m p l e t e l y decoupled spectrum is shown i n F i g . 4. The carbon c h e m i c a l s h i f t v a l u e s , shown i n T a b l e 3 a r e d e r i v ed from b o t h a d d i t i v i t y p r i n c i p l e s and t h e off-Resonance Spectrum F i g . 5 ( 5 ) .

CLOFI BRATE

203

Fig. 2.

Fig. 3.

in CDC13

W Spectrum of Clofibrate in Methanol.

PMR Spectrum of Clofibrate and Tetramethylsilane

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

204

F i g . 4.

Fig. CDC13

5.

I3C-NMR

13C-NMR

Spectrum of C l o f i b r a t e i n CDC13

Proton-Coupled

S p e c t r u m of C l o f i b r a t e i n

CLOFIBRATE

205

Table 3

13C NMR C h a r a c t e r i s t i c s of C l o f i b r a t e Carbon No.

2.54

Chemical S h i f t p pm

Carbon No.

Chemical S h i f t ppm

154.18

7

79.62

120.85

8

25.34

129.04

9

25.34

121.43

10

173.67

129.04

11

61.30

120.85

12

14.03

Mass Spectrum The mass spectrum of c l o f i b r a t e ( F i g . 6 ) o b t a i n e d by e l e c t r o n impact i o n i z a t i o n shows a molecular i o n M+ a t m / e 242 ( r e l a t i v e i n t e n s i t y 41.1%) and a b a s e peak a t 128. The proposed f r a g m e n t a t i o n p a t t e r n i s p r e s e n t e d i n T a b l e 4.

-

-

180

190

200 210

Fig. 6.

220

230

240 250

Mass Spectrum of C l o f i b r a t e .

260

270

280

290

300

207

CLOFIBRATE

Table 4 Mass F r a g m e n t a t i o n P a t t e r n of C l o f i b r a t e .

a

CB2

-

m/e

RI% -

Ion -

m/e -

R I%

Ion -

243

100

M.H

242

41.1

M+

169

27

a

169

33.1

a

129

10

-

130

33.0

b+2H

115

78

-

128

100.0

87

21.2

+

b

c+H

R I = Relative Intensity Mass s p e c t r a l d a t a , b o t h by e l e c t r o n i m p a c t and by m e t h a n e c h e m i c a l i o n i z a t i o n have a l s o been r e p o r t e d . ( 2 , 6, 7 ) .

3.

Synthesis: The s y n t h e s i s of C l o f i b r a t e h a s b e e n a c h i e v e d by two main methods (8-15). Method I : T h i s i n v o l v e s c o n d e n s a t i o n of p a r a - c h l o r o p h e n o l w i t h a c e t o n e and c h l o r o f o r m i n t h e p r e s e n c e o f sodium h y d r o x i d e f o l l o w e d by e s t e r i f i c a t i o n of t h e resulting acid, afforded clof ibrate.

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

208

Method I1 : By condensing phenol w i t h e t h y l 2-chloro-2methyl-propionate i n t h e p r e s e n c e of a s u i t a b l e dehydroc h l o r i n a t i n g a g e n t and t h e n c h l o r i n a t i o n of t h e r e s u l t a n t product, afforded c l o f i b r a t e .

C~G OH

+

CH3COCH 3

Clof i b r a t e

,or + OH

F-C00C2R5

cl- CH3

+

CHC13

CH3

I

dehydrochlor i n a t i n g agent

c12

COOC2H5 Clof i b r a t e

CLOFIBRATE 4.

209

Metabolism: The metabolism of c l o f i b r a t e and d i f f e r e n t c l o f i b r i c a c i d d e r i v a t i v e s i n s e v e r a l a n i m a l s p c e c i e s and i n man were reported (16-22). I t h a s been shown t h a t c l o f i b r a t e i s c o m p l e t e l y h y d r o l y s e d t o c l o f i b r i c a c i d (para-chlorophenoxy i s o b u t y r i c a c i d ) which is t h e n c o n j u g a t e d and e x c r e t e d . I n man t h e two c o n j u g a t e s of c l o f i b r i c a c i d found i n u r i n e were p r e s e n t i n p l a s m a . The h i g h e s t plasma c o n c e n t r a t i o n s of c l o f i b r i c a c i d m e t a b o l i t e s were g e n e r a l l y found i n pat i e n t s w i t h r e n a l d i s e a s e . S i n c e c l o f i b r a t e undergoes r a p i d h y d r o l y s i s i n v i v o and i n v i t r o , t h e r e s u l t i n g c l o f i b r i c a c i d i s persumed t o b e t h e a c t i v e d r u g . S t u d i e s i n v i v o and i n v i t r o w i t h c l o f i b r a t e and c l o f i b r i c a c i d i n d i c a t e t h a t t h e l a t t e r may e x e r t i t s e f f e c t by m u l t i p l e modes of a c t i o n ( 2 3 ) .

5.

Pharmacolopy:

(24-32)

Clof i b r a t e r e d u c e s e l e v a t e d t r i g l y c e r i d e and c h o l e s t e r o l c o n c e n t r a t i o n s i n serum; t h e e f f e c t on serum l i p o p r o t e i n s i s p a r t i c u l a r l y e v i d e n t on t h e v e r y l o w - d e n s i t y f r a c t i o n . When a d m i n i s t e r e d t o r a t s , i t c a u s e d a d e c r e a s e i n serum t r i g l y c e r i d e and a l t e r a t i o n s i n a d i p o s e t i s s u e u p t a k e and release of L i p i d s ( 3 3 ) . The b i o c h e m i c a l changes produced i n c l u d e a d e c r e a s e i n a d e n y l c y c l a s e a c t i v i t y , i n h i b i t i o n of acetyl-coenzyme. A carboxylase, i n h i b i t i o n of c h o l e s t e r o l and t r i g l y c e r i d e b i o s y n t h e s i s . The i n h i b i t i o n of h e p a t i c t r i g l y c e r i d e f o r m a t i o n i s a n e a r l y metab o l i c consequence of c l o f i b r a t e a d m i n i s t r a t i o n and p r e c e d e s t h e f a l l i n serum t r i g l y c e r i d e . I t s e f f e c t s on blood c o a g u l a b i l i t y s u g g e s t t h a t i t may r e d u c e t h e hypercoagulability frequently associated with a t h e r o s c l e r o s i s Uric a c i d c o n c e n t r a t i o n s , where e l e v a t e d , h a v e f r e q u e n t l y shown a t r a n s i e n t r e d u c t i o n , and t h e s h o r t e n i n g of t h e r e c a l c i f i e d C l o t t i n g - t i m e , which o c c u r s d u r i n g post-prand i a l lipacmia, i s prevented.

.

C l o f i b r a t e w a s f i r s t used i n c o n j u n c t i o n w i t h a n d e r o s t e r o n e b u t i t became e v i d e n t t h a t t h e e f f e c t s produced were n o t enhanced by t h e s t e r o i d . I t i s used i n a t h e r o s c l e r o t i c conditions manifested i n coronary h e a r t d i s e a s e and i n c e r e b r a l and v a s c u l a r d i s e a s e s , i n f a m i l i a l hyperc h o l e s t e r o l em i a and i n x a n thoma t o u s c o n d i t i o n s . E x u d a t i v e d i a b e t i c r e t i n o p a t h y h a s been improved by c l o f i b r a t e . Samuel e t a 1 ( 3 4 ) r e p o r t e d t h e s i g n i f i c a n t r e d u c t i o n of c h o l e s t e r o l l e v e l s by t h e combined o r a l a d m i n i s t r a t i o n of neomycin and c l o f i b r a t e . Musa e t a 1 ( 3 5 ) s t u d i e d t h e e f f e c t s of c l o f i b r a t e upon t h e d i s t r i b u t i o n , m e t a b o l i s m ,

MAHMOUD M. A. HASSAN AND AlDA A. ELAZZOUNY

210

t r a n s p o r t and plasma b i n d i n g of 1 3 1 1 - t h y r o x i n e i n e n t h y roid individuals. I t had no c o n s i s t e n t e f f e c t upon t h e b i n d i n g c a p a c i t i e s of t h y r o x i n e - b i n d i n g g l o b u l i n o r t h y r o x i n e b i n d i n g pre-albumin. The h e p a t i c d i s t r i b u t i o n s p a c e and c o n t e n t of t h y r o x i n e - i o d i n e were lower a f t e r c l o f i b r a t e . The h e p a t i c t h y r o x i n e c l e a r a n c e and plasmat o - l i v e r t h y r o x i n e f l u x were unchanged. C l o f i b r a t e d i d n o t a l t e r t h e plasma p y r o x i n e i o d i n e , t h e d a i l y t h y r o x i n e degradation r a t e o r t h e t o t a l thyroxine d i s t r i b u t i o n s p a c e . These f i n d i n g s f a i l e d t o s u p p o r t t h e h y p o t h e s i s t h a t c l o f i b r a t e p r o d u c e s i t s h y p o l i p i d e m i c e f f e c t by d i s p l a c i n g t h y r o x i n e from i t s b i n d i n g p r o t e i n s and s h u n t ing it i n t o t h e l i v e r . H a r r i s o n and Harden ( 3 6 ) s t u d i e d t h e e f f e c t of- c l o f i b r a t e on 6 p a t i e n t s w i t h h y p o t h y r o i d i s m and i s c h e m i c h e a r t d i s e a s e . The p a t i e n t s were m a i n t a i n e d on t h e maximum d o s e of t h y r o x i n e which t h e y c o u l d t o l e r a t e b u t a l l s t i l l had e v i d e n c e o f h y p o t h y r o i d i s m and l e v e l s of chol e s t e r o l i n t h e serum were e l e v a t e d . C l o f i b r a t e produced a r a p i d f a l l i n serum c h o l e s t e r o l a v e r a g i n g 37% and w a s m a i n t a i n e d up t o t h e two y e a r s t h e d r u g was c o n t i n u e d . I n t h r e e p a t i e n t s i t was p o s s i b l e t o i n c r e a s e t h e d o s e of t h y r o x i n e d u r i n g t h e r a p y w i t h c l o f i b r a t e ; i n two p a t i e n t s w i t h u n t r e a t e d s e v e r e h y p o t h y r o i d i s m , c l o f i b r a t e was w i t h o u t e f f e c t on serum c h o l e s t e r o l u n t i l t h y r o x i n e w a s added

.

I n vivo

Clof i b r a t e

Hydrolysis

-

Clof i b r i c a c i d Metabolism of Clof i b r a t e

Glucuron i d e

CLOFIBRATE

6.

21 1

Methods of A n a l y s i s 6.1

Elemental Analvsis

c,

59.38%

C 1 , 14.61:;

6.2

;

H,

;

0 , 19.78%.

6.232

;

I d e n t i f i c a t i o n Tests Those mentioned i n t h e B.P.

6.3

(1980) ( 3 7 ) .

A.

The i n f r a - r e d a b s o r p t i o n s p e c t r u m , Appendix I1 A , is c o n c o r d a n t w i t h t h e r e f e r e n c e s p e c t r u m of clof ibrate.

B.

The l i g h t a b s o r p t i o n , i n t h e r a n g e 220 t o 250nm, of a 2-cm l a y e r of a 0.001 per c e n t w/v s o l u t i o n i n a b s o l u t e e t h a n o l e x h i b i t s a maximum o n l y a t 226 nm; a b s o r b a n c e a t 226 nm, a b o u t 0.91, Append i x I1 B.

C.

The l i g h t a b s o r p t i o n , i n t h e r a n g e 250 t o 350 nm, of a 2-cm l a y e r of a 0.01 p e r c e n t w/v s o l u t i o n , i n a b s o l u t e e t h a n o l e x h i b i t s two maxima, a t 280 nm; and 238 nm a b s o r b a n c e a t 280 nm, aboilt 0.87, and a t 283 nm, a b o u t 0.62, Appendix I1 R .

D.

To 0.05 m l of a 1 0 per c e n t w/v s o l u t i o n i n e t h e r add 0.05 m l of a s a t u r a t e d s o l u t i o n of hydroxylammonium c h l o r i d e i n e t h a n o l (96 p e r c e n t ) and 0.05 m l of a s a t u r a t e d s o l u t i o n of potassium hydroxide i n e t h a n o l (96 p e r c e n t ) . Heat f o r two m i n u t e s on a w a t e r - b a t h , c o o l , a c i d i f y w i t h 0 . 5 M h y d r o c h l o r i c a c i d , and add 0.05 m l of a 1 p e r c e n t w/v s o l u t i o n of i r o n (111) c h l o r i d e ; a v i o l e t c o l o u r is produced.

P u r i t y Tests (a)

Water Content :

(b)

Acidity:

n o t more t h a n 0.2%.

Hix 1 0 . 0 g w i t h 1 0 0 m l o f n e u t r a l i z e d

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

212

a l c o h o l , add 3 d r o p s of p h e n o l p h t h a l e i n T.S. and t i t r a t e w i t h 0.1 N sodium h y d r o x i d e : n o t more t h a n 0 . 9 m l is r e q u i r e d f o r n e u t r a l i z a t i o n (c)

Para-Chlorophenol: The USP (XX) d e s c r i b e s a spectrophotometr i c method f o r t h e d e t e c t i o n of p-chlorophenol. The p e r c e n t a g e of p-chlorophenol i s n o t allowed t o exceed 0.003%, w h i l e t h e B.P. (1980) d e s c r i b e s a g a s chromatographic p r o c e d u r e .

6.4

O f f i c i a l Methods:

U.S.P. XX (38)

To a beaker c o n t a i n i n g 7 5 m l of 1 N sodium hydroxide add a b o u t 3 g of a s t r o n g l y b a s i c p o l y s t y r e n e anion-exchange r e s i n , and a l l o w t h e mixture t o stand f o r about 15 minutes, with o c c a s i o n a l s t i r r i n g . Wash t h e r e s i n w i t h water u n t i l t h e l a s t washing i s n e u t r a l t o l i t m u s paper, t h e n wash w i t h t h r e e 50 m l p o r t i o n s of methanol. Place a plug of g l a s s wool i n t h e b a s e of a 1-X 15 c m ion-exchange t u b e , and t r a n s f e r t o t h e t u b e a s u f f i c i e n t amount of Ion-exchange r e s i n , s l u r r i e d i n methanol, t o produce a column bed h e i g h t of from6-cm t o 8-cm. T r a n s f e r a b o u t 200 mg of C l o f i b r a t e , accur a t e l y weighed, t o a 100 m l v o l u m e t r i c f l a s k , add methanol t o volume, and mix. T r a n s f e r 10.0 m l of t h i s s o l u t i o n t o t h e Ion-exchange column, and c o l l e c t t h e e l u a t e i n a 100 m l v o l u m e t r i c f l a s k . R i n s e t h e column w i t h 25 m l of methanol, c o l l e c t t h e r i n s i n g i n t h e volumetric f l a s k , d i l u t e w i t h methanol t o volume, and mix. Transf e r 5 . 0 m l of t h i s s o l u t i o n t o a 50 m l volumet r i c f l a s k , d i l u t e w i t h methanol t o volume,and mix. D i s s o l v e a n a c c u r a t e l y weighed q u a n t i t y of USP C l o f i b r a t e RS i n methanol, and d i l u t e q u a n t i t a t i v e l y and s t e p w i s e w i t h methanol t o o b t a i n a s o l u t i o n having a known c o n c e n t r a t i o n of about 2 0 pg p e r m l . Concomitantly d e t e r m i n e t h e a b s o r h a n c e s of t h e Standard p r e p a r a t i o n and t h e Assay p r e p a r a -

213

CLOFIBRATE

t i o n i n 1-cm c e l l s a t t h e wavelength of maximum absorbance a t about 226 nm, w i t h a s u i t a b l e s p e c t r o p h o t o m e t e r , u s i n g methanol a s t h e b l a n k . C a l c u l a t e t h e q u a n t i t y , i n mg, of C12H15C103 i n t h e p o r t i o n of C l o f i b r a t e t a k e n by t h e formula lOC(Au/As), i n which C i s t h e c o n c e n t r a t i o n , i n ug per m l , of USP C l o f i b r a t e RS i n t h e Standard p r e p a r a t i o n , and Au and AS a r e t h e a b s o r b a n c e s of t h e Assay p r e p a r a t i o n and t h e Standard prepara t i o n , r e s p e c t i v e l y

.

6. 5 U l t r a v i o l e t Spectrophotometry A f t a l i o n e t a1 ( 3 9 ) r e p o r t e d t h a t e t h a n o l could n o t be used a s a s o l v e n t f o r t h e u l t r a v i o l e t s p e c t r o photometry of c l o f i b r a t e i n g e l a t i n o u s c a p s u l e s w i t h v e g e t a b l e o i l s because t h e l a t t e r i n t e r f e r e s t r o n g l y a t t h e Xmax of c l o f i b r a t e ( 2 2 7 nm). However, by using dioxane, t h e a b s o r p t i o n c o e f f i c i e n t of v e g e t a b l e o i l w a s found c o n s t a n t from 255 t o 280 nm w h i l e t h a t of c l o f i b r a t e i n c r e a s e d from 0 . 2 4 a t 265 nm t o 0.48 a t 280 nm ( u s i n g a 0.01% s o l u t i o n ) . I n t h e a s s a y , t h e c o n t e n t of 5 c a p s u l e s w a s homogenized, 0 . 5 g d i s solved i n dioxane t o g i v e 25 m l s o l u t i o n , 1 m l d i l u t ed f u r t h e r w i t h d i o x a n e t o 100 m l , and t h e a b s o r p t i o n w a s determined a t 280 nm and 265 nm w i t h r e s p e c t t o a s t a n d a r d s o l u t i o n of 0.01 g v e g e t a b l e o i l i n 100 m l dioxane measured a t 280 nm. The t r u e a b s o r p t i o n w a s taken a s t w i c e t h e d i f f e r e n c e a t t h e two wave l e n g t h s E r r o r s of ?4% were o b t a i n e d when a p p l i e d t o s y n t h e t i c m i x t u r e of 1 :1. A spectrophotometr i c a s s a y had been d e s c r i b e d ( 4 0 ) f o r t h e q u a n t i t a t i o n of c l o f i b r i c a c i d i n plasma and u r i n e . T h i s i n v o l v e s s o l v e n t e x t r a c t i o n of c l o f i b r i c a c i d from a c i d i f i e d plasma o r u r i n e and subsequent measurement of t h e UV absorbance a t 2 2 6 n m .

The U.S.P. X I X ( 4 1 ) d e s c r i b e s an a s s a y procedure f o r c l o f i b r a t e based on t h e p a s s a g e of t h e s o l u t i o n of c l o f i b r a t e i n methanol on a column of s t r o n g l y b a s i c p o l y s t y r e n e anion-exchange r e s i n . The a b s o r bances of t h e a s s a y p r e p a r a t i o n and a s t a n d a r d c l o f i b r a t e p r e p a r a t i o n are c o n c o m i t a n t l y determined i n 1-cm c e l l s a t 226 nm u s i n g methanol a s t h e blank.

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6.6

Thin Layer Chromatography (42) M i x t u r e s of c l o f i b r a t e and x a n t h i n o l n i c o t i n a t e were , s e p a r a t e d on s i l i c a g e l l a y e r s . Using c h l o r o form a s s o l v e n t , t h e hRf of c l o f i b r a t e and x a n t h i n o l n i c o t i n a t e a r e 82 and 3 r e s p e c t i v e l y . Using c h l o r o form a c e t i c a c i d 95:5, t h e hRf v a l u e s a r e : Clof i b r a t e ( 7 8 ) , x a n t h i n o l (0) and n i c o t i n i c a c i d (15) *

6.7

Thin Layer

- Gas L i q u i d Chromatography:

A s p e c i f i c and s e n s i t i v e method f o r t h e d e t e r m i n a t i o n of c l o f i b r a t e i n b i o l o g i c a l f l u i d s w a s d e s c r i bed ( 43 ) . C l o f i b r a t e w a s s e p a r a t e d from a s s o c i a t e d f a t t y a c i d s by TLC and t h e m e t h y l ester was q u a n t i f i e d by GLC.

6.8

Gas L i q u i d Chromatography: Gas l i q u i d c h r o m a t o g r a p h i c methods occupy a prominent p o s i t i o n i n t h e q u a n t i t a t i o n of c l o f i b r a t e i n d r u g s , t i s s u e s and b i o l o g i c a l f l u i d s . I n t h e method of S i l v e s t r i ( 4 4 ) , c l o f i b r a t e i s e x t r a c t e d from t a b l e t s w i t h e t h y l e t h e r and from t i s s u e s , blood o r u r i n e by homogenization w i t h t h e a d d i t i o n of N a C l and 7% HClO4 s o l u t i o n w i t h e t h e r - l i g h t p e t r o l e u m (1 :1). The e t h e r e x t r a c t is washed w i t h water, d r i e d o v e r anhydrous sodium s u l p h a t e and e v a p o r a t e d t o s m a l l volume i n a stream of n i t r o g e n . GLC performed a t 19OoC on a column ( 6 f t . x 1 / 8 i n . ) of 5% b u t a n e - d i o l s u c c i n a t e on Chromsorb P(100 t o 120 mesh), w i t h n i t r o g e n as c a r r i e r g a s and a f l a m e i o n i z a t i o n d e t e c t o r , methyl h e p t a d e c a n o a t e is used a s i n t e r n a l s t a n d a r d . Clof i b r i c a c i d i s s i m i l a r l y d e t e r m i n e d a f t e r a d s o r p t i o n on A m b e r l i t e IRA-400 and e s t e r i f i c a t i o n by treatment with methanolic HC1. Overall recoveries were 98X ( + 5 % ) f o r t h e ester and 92% ( + l O Z ) f o r t h e f r e e a c i d . The l i m i t of d e t e c t i o n i s 0 . 5 ug of t h e ester o r a c i d p e r m l of u r i n e o r g . of t i s s u e .

Karmen and Haut ( 4 5 ) d e s c r i b e d a method f o r a s s a y i n g c l o f i b r a t e i n serum based on GLC of t h e methyl e s t e r . Two i n t e r n a l s t a n d a r d s similar i n c h e m i c a l s t r u c t u r e t o c l o f i b r a t e (chlorophenoxy a c e t i c and chlorophenoxy p r o p i o n i c a c i d s ) a r e added; t h e compounds a r e e x t r a c t e d , c o n v e r t e d t o m e t h y l e s t e r s and s u b j e c t e d t o GLC u s i n g a n a l k a l i f l a m e i o n i z a t i o n

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215

d e t e c t o r s e l e c t i v e l y s e n s i t i v e t o halogen. Knuechel and Ochs ( 4 6 ) d e s c r i b e d a g a s chromatog r a p h i c method f o r t h e d e t e c t i o n of c l o f i b r a t e i n t h e serum of t r e a t e d p a t i e n t s . The r e t e n t i o n time of c l o f i b r a t e w a s found between m e t h y l p a l m i t a t e and m e t h y l stearate. C l o f i b r a t e was d e t e c t e d i n v e r y s m a l l amounts i n serum g l o b u l i n s which were s e p a r a t e d by f r a c t i o n a l p r e c i p i t a t i o n , w h e r e a s t h e amounts e x t r a c t e d from albumin c o r r e l a t e d w e l l w i t h t h o s e of t h e serum. F r a c t i o n s o f c h o l e s t e r o l ester from serum of c l o f i b r a t e - t r e a t e d p a t i e n t s , c o n t a i n e d s m a l l amounts of c l o f i b r a t e i n s p i t e of r e p e a t e d f r a c t i o n a t i o n on s i l i c a g e l column, d u e t o e s t e r i f i c a t i o n of c l o f i b r a t e by cholesterol. Berlin ( 4 7 ) reported a q u a n t i t a t i v e gas c h r o m a t e g r a p h i c method of c l o f i b r i c a c i d i n plasma u s i n g 2-(4chloro-3-methylphenoxy)-2-methylpropionic a c i d as int e r n a l s t a n d a r d . To blood plasma (200 p l ) i s added H3PO4 a c i d 2M (50 p l ) and i n t e r n a l s t a n d a r d s o l u t i o n in t o l u e n e (0.5 m l ) . A f t e r s h a k i n g (10 min) t h e o r g a n i c phase i s t r a n s f e r r e d t o a 3 m l t a p e r e d g l a s s t u b e and t h e plasma i s r e - e x t r a c t e d w i t h 0 . 5 m l t o l u e n e . Disodium hydrogen p h o s p h a t e 0.5M ( 0 . 5 ml) i s added t o t h e combined o r g a n i c p h a s e s and t h e m i x t u r e i s s h a k e n f o r 1 0 min. The o r g a n i c p h a s e i s removed and t h e aqueous phase i s made a c i d i c w i t h H3PO4 a c i d 5 M (50 p l ) CH2C12 (100 1-11>i s added and t h e m i x t u r e i s e x t r a c t e d on a whirl-mixer f o r 30 s e c o n d s . A f t e r removal of t h e aqueous p h a s e , diazomethane i n e t h e r (50 1-11>is added and t h e s o l u t i o n i s e v a p o r a t e d under n i t r o g e n t o a b o u t 1 / 5 of i t s volume and one p l i s i n j e c t e d . Blank plasma samples showed no p e a k s i n t e r f e r i n g w i t h t h e c l o f i b r i c a c i d o r i n t e r n a l s t a n d a r d m e t h y l ester p e a k s . The method i s l i m i t e d t o d e t e r m i n a t i o n of plasma c o n c e n t r a t i o n s down t o 1 pg ml-I d u e t o t h e c o n c e n t r a t i o n chosen f o r t h e i n t e r n a l s t a n d a r d . Another method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of c l o f i b r i c a c i d i n blood p l a s m a had been d e s c r i b e d ( 45 ) . The s u b s t a n c e is e x t r a c t e d from a c i d i f i e d plasma i n t o benzene, t h e e x t r a c t i s e v a p o r a t e d t o d r y n e s s and t h e r e s i d u e i s m e t h y l a t e d and s u b m i t t e d t o chromatography on a g l a s s column packed w i t h 3%O V 4 7 on chromosorb WHP, 100-120 mesh. The c o n d i t i o n s a r e a s f o l l o w s : i n j e c t o r t e m p e r a t u r e , 18OoC; d e t e c t o r

216

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY t e m p e r a t u r e , 210°; column i n i t i a l t e m p e r a t u r e , 150° f o r 6 min. t h e n programmed a t 195' a t 10°/min. and h e l d a t t h e f i n a l t e m p e r a t u r e f o r 5 min. b e f o r e rec y c l e . The f l o w r a t e s were: helium c a r r i e r g a s , 25 ml/min. helium a u x i l i a r y g a s , 35 ml/min; a i r , 400 m l / min. hydrogen, 40 m l / m i n . The r e t e n t i o n t i m e s of c l o f i b r i c a c i d m e t h y l ester and of a c t a d e c a n e ( e x t e r n a l s t a n d a r d ) were 3 and 5 min. r e s p e c t i v e l y . A r a p i d g a s c h r o m a t o g r a p h i c method i s d e s c r i b e d

( 4 9 ) f o r t h e d e t e r m i n a t i o n of c l o f i b r i c a c i d i n p l a s m a and u r i n e . The a s s a y i n v o l v e s a n e x t r a c t i o n i n t o t o l u e n e and b a c k - e x t r a c t i o n of c l o f i b r i c a c i d and t h e i n t e r n a l s t a n d a r d ( 2 - n a p h t h o i c a c i d ) i n t o t h e methylating agent (trimethylanilinium hydroxide). The s i l a n i z e d g l a s s column ( 6 f t . x 4 mm i . d . ) was packed w i t h 3% of SE-30 on 80-100 mesh Gas Chrom Q 0 and was o p e r a t e d a t 150 C w i t h a c a r r i e r g a s ( n i t r o gen) f l o w - r a t e o f 25 ml/min ; t h e i n j e c t i o n p o r t temp e r a t u r e w a s 29OoC. The f l a m e i o n i z a t i o n d e t e c t o r was o p e r a t e d a t 27OoC w i t h a hydrogen f l o w - r a t e of 20 mllmin. and a n oxygen f l o w - r a t e of 200 mllmin. Under t h e s e c o n d i t i o n s , t h e r e t e n t i o n times were 1 . 5 min. f o r c l o f i b r i c a c i d and 2 . 5 min. f o r t h e i n t e r n a l s t a n d a r d . The blood samples a r e drawn i n t o h e p a r i n i zed t u b e s and t h e plasma w a s s e p a r a t e d by c e n t r i f u g a t i o n . To 1 . 0 m l of plasma i n a 1 5 m l g l a s s t u b e were added 1 m l of 0.4 M h y d r o c h l o r i c a c i d and 6 m l o f t o l u e n e c o n t a i n i n g 120 pg of t h e i n t e r n a l s t a n d a r d . The t u b e w a s shaken f o r 5 min. and c e n t r i f u g e d f o r 3 min. a t 4000 g . A 5 m l p o r t i o n of t h e o r g a n i c phase i s t r a n s f e r r e d t o a p o i n t e d c e n t r i f u g e t u b e . T r i m e t h y l a n i l i n i u m h y d r o x i d e ( 5 0 p l ) i s added and t h e m i x t u r e i s e x t r a c t e d on a Vortex-mixer f o r 1 min. A f t e r b r i e f c e n t r i f u g a t i o n , 1 p 1 o f . t h e aqueous layer is injected directly. The d e t e r m i n a t i o n of u r i n a r y c l o f i b r i c a c i d is c a r r i e d o u t e s s e n t i a l l y a s d e s c r i b e d f o r plasma. For a n a l y s i s of g l u c u r o n i d e m e t a b o l i t e of c l o f i b r i c a c i d i n u r i n e , t h e sample i s d i l u t e d 1 : l O w i t h 0.2 M sodium a c e t a t e b u f f e r of pH 5.0 and 4 ml of t h e d i l u t e d sample are i n c u b a t e d o v e r n i g h t w i t h 2000 Fishman u n i t s of a g l u c u r o n i d a s e a r y l - s u l p h a t a s e p r e p a r a t i o n from R e l i x Pomatia.

Wolf and Zimmerman ( 5 0 ) d e s c r i b e d a sumultaneous GLC d e t e r m i n a t i o n of C l o f i b r a t e and i t s m e t a b o l i t e c l o f i b r i c a c i d i n human plasma. T h i s h a s been achi e v e d by u s i n g a g a s c h r o m a t o g r a p h i c column packed

217

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w i t h Gas Chrom Q c o a t e d w i t h 10% S i l a r 1 0 C and n i t r o g e n a s c a r r i e r g a s . The method is r a p i d and do n o t require a derivatization step, it is sensitive to 1 u g / d of e i t h e r compound i n b i o l o g i c a l s a m p l e s and c o u l d b e used t o c h a r a c t e r i z e t h e i n v i v o c o n v e r s i o n of c l o f i b r a t e ester t o t h e f r e e a c i d . A c o m p a r a t i v e s t u d y of g a s - l i q u i d c h r o m a t o g r a p h i c b e h a v i o u r of t h e p e n t a f l u o r o b e n z y l esters and t h e m e t h y l esters of t e n c h l o r o p h e n o x y a l k y l a c i d s i n c l u d i n g c l o f i b r i c a c i d was a l s o r e p o r t e d ( 5 1 ) P a r a - c h l o r o p h e n o l and para-hydroxy b e n z o i c a c i d esters which are added a s c a p s u l e p r e s e r v a t i v e s were d e t e r mined by g a s chromatography ( 52 ) . T h e s e were rea c t e d w i t h (EtO)2 P ( 0 ) C l and MeONa i n h e x a n e a t 50° f o r 3 0 min. t o g i v e t h e d i e t h y l p h o s p h a t e esters. These esters and c l o f i b r a t e were s e p a r a t e d and d e t e r mined on a column packed w i t h 3 . 5 % s i l i c o n e J X R on Chromosorb G and HP a t 190' u n d e r N2 c a r r i e r . Flame p h o t o m e t r i c and Flame t h e r m i o n i c d e t e c t o r s were u s e d . The c o e f f i c i e n t s of v a r i a t i o n f o r 3 . 8 7 , 7.74 pg p a r a c h l o r o p h e n o l i n 1 0 m l were 0 . 8 7 , 0.56% w i t h FTB and 1.5, 0.56% w i t h FPD r e s p e c t i v e l y . P a r a c h l o r o p h e n o l i n commercial p r e p a r a t i o n s was 1.6-5.1 ppm.

6.9

Gas Chromatography

- Mass S p e c t r o m e t r y :

I m p u r i t i e s i n c l o f i b r a t e h a v e been s t u d i e d GC-MS T h r e e main i m p u r i t i e s were f o u n d , t h e m e t h y l ester a n a l o g u e of c l o f i b r a t e , i t s d e s c h l o r o a n a l o g u e and t h e d i c h l o r o a n a l o g u e . ( 53 ) .

J o h a n s s o n and Ryhage ( 5 4 ) have i d e n t i f i e d o t h e r i m p u r i t i e s i n t h r e e c l o f i b r a t e p r e p a r a t i o n s . Samples were o b t a i n e d by d i s s o l v i n g 0 . 5 m l of c a p s u l e - cont e n t i n 0.5 m l c h l o r o f o r m 5 p 1 of t h e s a m p l e w a s i n j e c t e d i n t o t h e combined g a s c h r o m a t o g r a p h - mass s p e c t r o m e t e r LKB 2091. The G . C . column u s e d w a s 3% A constant SE-30 g l a s s column 2.7 M x 2 mm ( i . d . ) . t e m p e r a t u r e of 16OoC f o r t h e f i r s t 8 min. w a s u s e d . The c a r r i e r g a s f l o w - r a t e was 25 m l h e l i u m l m i n . The mass s p e c t r a were o b t a i n e d w i t h a c o n s t a n t accelerati n g v o l t a g e of 3 . 5 KV, a n e l e c t r o n e n e r g y of 70 eV and a n i o n i z i n g c u r r e n t of 100 PA. R e p e t i t i v e scann i n g of t h e m a s s r a n g e 1 0 t o 500 i n 25 w a s u s e d .

218

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY 6.10 High-Performance Liquid Chromatography: Bjornsson e t a 1 (55 ) developed a r a p i d , s e n s i t i v e and s p e c i f i c h i g h p r e s s u r e l i q u i d chromatograp h i c method f o r t h e q u a n t i t a t i v e a n a l y s i s of c l o f i b r i c a c i d i n plasma, s a l i v a and u r i n e . Plasma (0.1 -1 m l ) , s a l i v a (1 ml) o r u r i n e d i l u t e d 1 : l O O w i t h d i s t i l l e d water ( 1 . 0 ml) is p l a c e d i n a screw-capped t u b e , and 100 p 1 of i n t e r n a l s t a n d a r d s o l u t i o n (cont a i n i n g 6.7 pg of t h e i n t e r n a l s t a n d a r d ) , 0.5 m l of 0 . 5 N s u l p h u r i c a c i d and 5 m l of t o l u e n e a r e added. The samples a r e e x t r a c t e d by mixing f o r 10 min. f o l lowed by c e n t r i f u g a t i o n a t 1200 g f o r 10 min. t o s e p a r a t e t h e o r g a n i c and aqueous p h a s e s . The lower aqueous phase i s f r o z e n by immersing t h e t u b e i n a d r y - i c e a c e t o n e b a t h , and t h e o r g a n i c p h a s e i s poured i n t o a n o t h e r t u b e , which h a s a n e l o n g a t e d cone a t i t s b a s e . Then 50 p 1 of 0 . 2 N NaOH are added, and t h e m i x t u r e i s e x t r a c t e d on a Vortex-mixer f o r 2 rnin. A f t e r b r i e f c e n t r i f u g a t i o n , t h e aqueous phase i s drawn i n t o a s y r i n g e t h a t a l r e a d y c o n t a i n s 10 p 1 of a s o l u t i o n of 5% g l a c i a l a c e t i c a c i d i n water and t h i s m i x t u r e i s i n j e c t e d i n t o t h e chromatograph. For t h e a n a l y s i s of t h e g l u c u r o n i d e c o n j u g a t e of c l o f i b r i c a c i d i n u r i n e , 5 m l of 6N h y d r o c h l o r i c a c i d were added t o each sample, and t h e s o l u t i o n s were h e a t e d a t 98OC f o r 30 min. b e f o r e t h e e x t r a c t i o n . The samples were t h e n cooled and a n a l y s e d as d e s c r i b e d above, e x c e p t t h a t t h e a d d i t i o n of d i l u t e s u l p h u r i c a c i d i n t h e f i r s t s t e p w a s o m i t t e d . A V a r i a n Micropak CH-10 r e v e r s e - p h a s e column ( 2 5 c m x 6 . 3 mm 0.d. x 2.2 mm i . d . ) was used. The a b s o r b a n c e w a s measured a t 235 11111. One pump of t h e dual-pump g r a d i e n t - e l u t i o n chromatograph c o n t a i n e d a c e t o n i t r i l e and t h e o t h e r 0.5% a c e t i c a c i d i n d i s t i l l e d water; an i s o c r a t i c 42% a c e t o n i t r i t e m i x t u r e of t h e two s o l v e n t s was used. The f l o w - r a t e of t h e s o l v e n t m i x t u r e w a s 70 me/hr w i t h a column i n p u t p r e s s u r e of 197 a t m (2900 p.s.i.). C o n c e n t r a t i o n s between 1 . 0 and 25 pg p e r sample could be measured w i t h a c o e f f i c i e n t of v a r i a t i o n from 1-6%. 40-50 samples c a n e a s i l y be a s s a y e d i n a day. The method d o e s n o t r e q u i r e p r i o r chromatographic preparation o r m u l t i p l e extractions. Woodhouse e t a1 ( 5 6 ) d e s c r i b e d a high-performance l i q u i d chromatographic method f o r measuring plasma c o n c e n t r a t i o n s of c l o f i b r i c a c i d a f t e r administ r a t i o n of c l o f i b r a t e t o humans. 50 pg of t h e i n t e r -

CLOFIBRATE

219

n a l s t a n d a r d (4-chloro-2-methylphenoxyacetic a c i d ) i n methanol and 3M H C 1 (0.5 ml) were added t o plasma (1 ml) i n a g l a s s s t o p p e r e d c e n t r i f u g e t u b e , s h a k e n , allowed t o s t a n d f o r 5 min. and t h e n e x t r a c t e d w i t h 6 m l e t h e r . Ether w a s evaporated t o d r y n e s s under n i t r o g e n and t h e r e s i d u e d i s s o l v e d i n m e t h a n o l . 10 1~1p o r t i o n s of t h i s s o l u t i o n were i n j e c t e d i n t o t h e chromatograph u s i n g a s t o p - f l o w i n j e c t i o n t e c h n i q u e . The s t a i n l e s s s t e e l column (25 x 0.46 c m i . d . ) w a s packed w i t h c18 P a r t i s i l (10 pm). The m o b i l e p h a s e w a s 27% a c e t o n i t r i l e c o n t a i n i n g 0.4% o r t h o p h o s p h a t e b u f f e r ( t o m a i n t a i n t h e pH a t 4.2) a t a f l o w - r a t e of 2 mlfmin and a b a c k p r e s s u r e of 50 b a r . R e t e n t i o n times of i n t e r n a l s t a n d a r d and c l o f i b r i c a c i d were 6 and 7 min. r e s p e c t i v e l y . 7,

P r o t o n Magnetic Resonance S p e c t r o m e t r y : Hassan and L o u t f y ( 4 ) h a v e d e v e l o p e d PMR a n a l y t i c a l method f o r t h e q u a n t i t a t i o n of c l o f i b r a t e a s a d r u g e n t i t y and i n c a p s u l e d o s a g e form. The f o u r a r o m a t i c p r o t o n s q u a r t e t c e n t r e d a t 7 . 0 3 ppm ( F i g . 3) was chosen f o r q u a n t i t a t i o n of c l o f i b r a t e . Malonic a c i d w a s employed a s a n i n t e r n a l s t a n d a r d , s i n c e i t e x h i b i t s two p r o t o n s m e t h y l e n e s i n g l e t a t 3.36 ppm ( F i g . 7) which i s w i d e l y s e p a r a t e d from t h o s e of c l o f i b r a t e . Acetone was used a s t h e s o l v e n t , s i n c e c l o f i b r a t e and m a l o n i c a c i d a r e s o l u b l e i n i t and i t s m e t h y l p r o t o n s s i n g l e t a t 2.07 ppm ( F i g . 7 ) d o e s n o t i n t e r f e r e w i t h t h e d o w n f i e l d p r o t o n s of b o t h compounds. The p r o c e d u r e proved t o b e s i m p l e , r a p i d , a c c u r a t e and p r e c i s e . S t a n d a r d d e v i a t i o n s of *1.07% and +1.34% were o b t a i n e d f o r p u r e d r u g and c a p s u l e s r e s p e c t i v e l y . The PMR spectrum i n a d d i t i o n , p r o v i d e s a s p e c i f i c means of i d e n t i f i c a t i o n of c l o f i b r a t e , and d e t e c t i o n of i m p u r i t i e s . Another PMR p r o c e d u r e have been a l s o r e p o r t e d For t h e b u l k d r u g , 100 t o 150 mg of sample i s shaken w i t h 5 m l of a s o l n . (10 mg ml-I of t h e i n t e r n a l s t a n d a r d (hexamethylcyclotrisilazane) i n CCl4 and t h e m i x t u r e is t r a n s f e r r e d t o a NMR t u b e . For c a p s u l e s , t h e c o n t e n t s a r e e x t r a c t e d w i t h C C l 4 ( 4 x 5 m l ) , t h e combined e x t r a c t s a r e made up t o 25 m l w i t h CCl4 and 5 m l of t h i s s o l n . i s p l a c e d i n t h e n.m.r. t u b e , t o g e t h e r w i t h 1 m l of t h e i n t e r n a l s t a n d a r d s o l n . (40 t o 50 mg m l - I ) . The NMR spectrum i s t h e n

(57 1 f o r t h e d e t e r m i n a t i o n of c l o f i b r a t e .

220 5

. . . . l . . . . , . . . . , . . . . l . , . . , . . .

.

I

MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY

600

I

'

,JyI.118.0

I

3OQ

400

70

I

2M

'

I 6.0

5.0 P H ( 6 ) 4 0

PARTS RFU MILLION, 6

F i g . 7 . PMR Spectrum of C l o f i b r a t e , Malonic a c i d and T e t r a m e t h y l s i l a n e i n Acetone.

r e c o r d e d , and t h e c l o f i b r a t e concn. i s c a l c u l a t e d by comparing t h e i n t e g r a l of t h e s i n g l e t peak a t 1 . 4 7 p.p.m. f o r c l o f i b r a t e w i t h t h a t f o r t h e i n t e r n a l s t a n d a r d . The p r o c e d u r e i s s i m p l e , r a p i d and accur a t e , and t h e r e c o v e r y i s 9 9 . 6 ? 3 . 2 0 X . Acknowledgement : The a u t h o r s would l i k e t o t h a n k Elr. T a n v i r A . B u t t o f t h e Department of P h a r m a c e u t i c a l C h e m i s t r y , C o l l e g e of Pharmacy, King Saud U n i v e r s i t y , f o r t y p i n g t h e m a n u s c r i p t .

22 1

CLOFIBRATE 9.

References:

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Aboul-Enein,

Spec. L e t t . ,

11

John G . Hoogerhekk and Bruce E . Wyka

1. Description 1.1 Names, Formula, Molecular Weight, and Structure 1.2 Drug Properties 1.3 Appearance, Color, Odor, and Taste 2. Physical Properties 2.1 Nuclear Magnetic Resonance Spectra 2.2 Mass Spectrum 2.3 Infrared Spectrum 2.4 Ultraviolet Spectrum 2.5 Melting Range 2.6 Thermal Properties 2.7 Crystal Properties 2.8 Solubility 2.9 Dissociation Constant 3. Synthesis 4. Stability 5 . Drug Metabolism and Pharmacokinetics 5.1 Drug Metabolism 5.2 Pharmacokinetics 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Identification 6.3 Spectrophotometric Analysis 6.4 Titrimetric Analysis 6.5 Chromatographic Analysis 6.6 Radiochemical Analysis 6.7 Microcalorimetric Analysis 6.8 Microbiological Analysis 7. Acknowledgements 8. References

Analytical Profiles of Drug Substances Volume 11

225

226 226 226 226 227 227 229 232 232 235 235 236 241 241 242 242 244 244 244 246 246 247 247 248 248 25 1 252 252 252 253

Copyright 0 1982 by The American Pharmaceutical Association ISBN 0-12-260811-9

JOHN G. HOOGERHEIDEAND BRUCE E. WYKA

226

1.

Description

1.1 Names, Formula, M o l e c u l a r Weight

and S t r u c t u r e

C l o t r i m a z o l e was f i r s t s y n t h e s i z e d i n 1969 b y Plempel e t a l . (1) and t e s t e d under t h e name Bay b5097. The compound-iias been marketed w i d e l y under t h e t r a d e names o f Canesten, L o t r i m i n , Gyne-Lotr imin, and Mycelex. The chemical name o f c l o t r i m a z o l e i s 1 - ( 2 - c h l o r o -

pheny1)diphenylmethyl-1H- imidazole.

The m o l e c u l a r f o r m u l a o f c l o t r i m a z o l e i s C 2 2 H i 7 C l N z and t h e m o l e c u l a r weight i s 344.8 g/mol. The s t r u c t u r e i s g i v e n i n F i g u r e 1. 1.2 Drug P r o p e r t i e s C l o t r i m a z o l e i s a broad-spectrum a n t i m y c o t i c agent e f f e c t i v e a g a i n s t pathogenic dermatophytes, yeasts, and s e v e r a l species o f Candida, T r i c h o hyton, Microsporum, Epidermophyton, and Maa-i. reparations o f the drug are used b o t h i n t h e t o p i c a l t r e a t m e n t o f dermal i n f e c t i o n s and t o combat v u l v o v a g i n a l c a n d i d i a s i s .

&

Results o f i n i t i a l c l i n i c a l studies w i t h t h i s compound p u b l i s h e d i n 1969 ( 2 ) were f o l l o w e d b y more d e t a i l e d r e p o r t s o u t l i n i n g t h e spectrum and mechanisms o f At therapeutic clotrimazole activity (1,3,4). c o n c e n t r a t i o n s d r u g a c t i o n i s f u n g i s t a t i c ; however, a t h i g h c o n c e n t r a t i o n s ( 2 0 p g / m l ) some invitro fungicidical a c t i v i t y has been observed (1,4). 1.3 Appearance, C o l o r , Odor and T a s t e Clotrimazole i s a colorless, c r y s t a l 1i n e s o l i d .

odorless,

tasteless,

227

CLOTRIMAZOLE

2.

Physical Properties

2.1

Nuclear Magnetic Resonance S p e c t r a

2.1.1

P r o t o n Magnetic Resonance

The p r o t o n NMR spectrum ( F i g u r e 2) o f a 10% ( w / v ) solution o f clotrimazole i n deuterated chloroform a t ambient temperature was o b t a i n e d b y u s i n g a V a r i a n CFT-20 spectrometer o p e r a t i n g a t a frequency o f 79.5 MHz. The chemical s h i f t s g i v e n i n Table I a r e d o w n f i e l d from t h e internal reference tetramethylsilane. P r o t o n assignments are as i n d i c a t e d i n F i g u r e 1. A 220 MHz p r o t o n NMR spectrum o f a s o l u t i o n o f c l o t r i m a z o l e i n C 6 D 6 has a l s o been r e p o r t e d i n t h e 1i t e r a t u r e ( 5 ) . Table I PMR S p e c m s i g n r n e n t s

Protons

Chemical S h i f t s (6)

Intensity

Multiplicity

5-H

6.75

1H

triplet,

4-H

7.00

1H

triplet, J=1.5 Hz

2-H

7.40

1H

triplet,

3'-H t o 6 ' - H 2"-H t o 6"-H 8"-H t o 12"-H

6.95-7.40

14H

J=1.5 Hz

J=1.5 Hz

broad mu1t i p l e t s

228

JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

10"

F i g u r e 1. S t r u c t u r e of C l o t r i m a z o l e .

I l

"

1

9

I "

'

"

I

8

1

I

7

6

I

I

I

I

I

I

L

4

3

2

1

0

I

s 5 8H

79.5 MHz

F i g u r e 2. Proton N u c l e a r Magnetic R e s o n a n c e S p e c t r u m of C l o t r i m a z o l e in CDC13.

229

CLOTRIMAZOLE

2.1.2

Carbon-13 Magnetic Resonance

The carbon-13 p r o t o n decoupled NMR spectrum ( F i g u r e 3 ) o f a 20% ( w / v ) s o l u t i o n o f c l o t r i m a z o l e i n d e u t e r a t e d c h l o r o f o r m a t ambient t e m p e r a t u r e was o b t a i n e d b y u s i n g a V a r i a n XL-100 s p e c t r o m e t e r o p e r a t i n g a t a f r e q u e n c y o f 25.2 MHz. The c h e m i c a l s h i f t s a r e i n ppm ( 6 ) w i t h r e f e r e n c e t o internal tetramethylsilane. The carbon-13 NMR spectrum i n d i c a t e s t h e presence o f f i v e q u a r t e r n a r y carbons a t 6 = 75.0, 135.6, 139.1, 140.4, and 140.9, and seventeen o l e f i n i c carbons a t 6 = 121.5, 127.0, 128.0 (4 e q u i v a l e n t c a r b o n s ) , 128.1 ( 3 e q u i v a l e n t c a r b o n s ) , 128.5, 129.8, 130.1 ( 4 e q u i v a l e n t c a r b o n s ) , 130.4, and 132.2 ppm. 2.2

Mass Soectrum

The mass spectrum ( F i g u r e 4 ) o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g a V a r i a n MAT CH5 medium r e s o l u t i o n s i n g l e focusing instrument. The e l e c t r o n e n e r g y was 70 eJ, and t h e probe and source t e m p e r a t u r e s used were 140 C and 25OoC, r e s p e c t i v e l y . The r e s u l t s a r e g i v e n i n T a b l e 11. Table I1

m/e

Ions -

344

M'

309

(M-35)'

277

(M-67)'

242

(M-102)'

241

(M-103)'

239

(M-105)'

199

c) 165

*C3H3N2

=

t H5C6CC6H 4

1111

4r"' ' '

I

'

8

' '~

IIY)

" ' " " ' '

Figure 3 .

a

!

I

I40 ' " " ' ' , l ' ' '

;':

'3-0 1 1 ; '

190

I , , , ,

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2

, , I ,

' , ~ ' ~ , ' ' , , , , , , . l , , , , , , , , . I

#

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# I , ,

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I

I 1

I

I

,,,,,

, , , / I ,

. i l

,

,

, * , oI , , ,

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18. v v m l

Carbon-13 Nuclear Magnetic Resonance Spectrum o f Clotrimazole in CDC13.

20 I

I

P

7

0

z

A 1 SN31NI 3AllWl3U 23 1

0

VI

m

0 O

m

0

In (u

v)

0 0 : O W

I

z

3 v) v)

- I

:a

JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

232

2.3

I n f r a r e d Spectrum

The i n f r a r e d spectrum ( F i g u r e 5 ) o f c l o t r i m a z o l e as a d i s p e r s i o n i n m i n e r a l o i l was o b t a i n e d b y u s i n g a P e r k i n Elmer Model 180 i n f r a r e d spectrophotometer The major a b s o r p t i o n bands a r e g i v e n i n Table 111.

.

The i n f r a r e d s p e c t r u m o f c l o t r i m a z o l e as a d i s p e r s i o n i n potassium bromide has been r e p o r t e d i n t h e 1i t e r a t u r e ( 5 ) . Table I 1 1 I n f r a r e d Band Assignments Wavenumber ( c m - l )

Assignment

3170, 3115, 3085, 3075 (w)

aromatic C-H ( s t r e t c h )

1585, 1570 (w)

aromatic C=C, C=N (stretch)

1510, 1500, 1450 (m)

a r o m a t i c C=C, C=N (stretch)

770, 760, 750 ( v s )

aromatic C-H bend

720, 700, 680 ( S - V S )

aromatic C-H o u t - o f - p l ane bend

Notations: w = weak 2.4

vs = very strong;

s = strong;

o u t - o f - p l ane

m = medium;

U l t r a v i o l e t Spectrum

The u l t r a v i o l e t s p e c t r a o f c l o t r i m a z o l e i n methanol and methanol i c 0.1N h y d r o c h l o r i c a c i d ( F i g u r e 6 ) were o b t a i n e d b y u s i n g a C a G Model 118 spectrophotometer. The maxima, m i n i m a , s h o u l d e r s and r e s p e c t i v e m o l a r a b s o r p t i v i t i e s are g i v e n i n Table I V .

2.5

Figure 5.

3

4

WAVELENGTH 5 6

7

MICRONS 8 9 10

I n f r a r e d Spectrum o f C l o t r i m a z o l e i n Mineral O i l .

12 14

1822

3550

JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

234

250

Figure 6 .

300 WAVELENGTH (nm)

Ultraviolet Spectra o f Clotrimazole.

350

235

CLOTRIMAZOLE

Table I V U 1t r av io 1e t Spectra 1 Character is t ics o f C 1o t r imazo 1e

Solvent

A (nm)

Methanol

274 269 265 263 259 253 249

(shoulder) (shoulder) (shoulder) (shoulder) (maximum) (maximum) (minimum)

1.03 2.40 3.32 3.21 3.40 2.94 2.64

Methanol i c 0.1N hydrochloric acid

274 269 263 261 259 254 246

(shoulder) (shoulder) (maximum) (minimum) (maximum) (shoulder) (minimum)

1.75 3.28 3.96 3.84 3.93 3.12 2.38

2.5

E

x 10-

2

M e l t i n g Range The f o l l o w i n g me1 t i n g ranges have been r e p o r t e d :

2.6

M e l t i n g Range

Reference

143 t o 144OC

596

144 t o 145OC

7

141 t o 145OC

a

Thermal P r o o e r t i e s 2.6.1

D i f f e r e n t ia1 Scann i n g C a l o r i m e t r y

The d i f f e r e n t i a l scanning c a l o r i m e t r y c u r v e ( F i g u r e 7 ) o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g oa DuPont Model 990 Thermal Analyzer a t a h e a t i n g r a t e o f 1 0 C/minute under a n i t r o g e n atmosphere. A s i n g l e sharp endotherm was observed w i t h an e x t r a p o l a t e d onset temperature o f 143OC.

JOHN G. HOOGERHEIDEAND BRUCE E. WYKA

236

P u r i t y a n a l y s i s b y d i f f e r e n t i a l scanning c a l o r i ( F i g u r e 8 ) was p e r f o r m e d a t a h e a t i n g r a t e o f 1 C/minute under a n i t r o g e n atmosphere. The p u r i t y o f t h e sample was d e t e r m i n e d t o b e 99.5 m o l e p e r c e n t . The l a t e n t h e a t o f f u s i o n AH^) was c a l c u l a t e d t o be 7540 c a l / m o l .

mgtry

2.6.2

Thermograv i m e t r y

Thermogravimetric a n a l y s i s ( F i g u r e 9 ) o f c l o t r i mazole was p e r f o r m e d b y u s i n g a DuPont Modelo950 Thermog r a v i m e t r i c A n a l y z e r a t a h e a t i n g r a t e o f 10 C/minute under No weighto loss was observed f r o m a n i t r o g e n atmosphere. ambient temper,ature t o about 180 C. The g r a d u a l w e i g h t loss above 180 C i s due t o v a p o r i z a t i o n o f t h e m e l t . 2.7

Crystal Properties 2.7.1

X-Rav D i f f r a c t i o n

The X-ray powder d i f f r a c t i o n p a t t e r n ( F i g u r e 10) o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g a P h i l l i p s DP-3500 X - r a y D i f f r a c t o m e t e r and Cu K, r a d i a t i o n (1.5148 ) . The d a t a a r e g i v e n i n T a b l e V.

if

2.7.2

P o l ymorph ism

Borka e t a l . ( 6 ) have r e p o r t e d t h e f o r m a t i o n o f a f clotrimazole from a cr%stal f i l m metastable f o f i o p r e p a r a t i o n . They r e p o r t a m e l t i n g p o i n t o f 106 C f o r t h i s m e t a s t a b l e f o r m and an i n f r a r e d spectrum t h a t i s d i f f e r e n t from t h e s t a b l e form.

CLOTRIMAZOLE

!37

'0 TEMPERATURE.

"c

F i g . 7. D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y Curve of Clotrimazole.

TEMPERATURE, *K

F i g . 8. D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y Curve of C l o t r i mazole f o r P u r i t y A n a l y s i s .

238

W 0

7

N

E

a L

.r

0

+J 0

c

le 0)

0

3

L

w 1,

0

L c, a, E

l-0

5

.I-

L

rn 0

E L a,

L: I-

0,

W L S

m L L

*I-

1

00 7

01

40

I

36

I

32

Figure 10.

1

20

i

1

24

20

1

16

1

12

28

Powder X-ray D i f f r a c t i o n P a t t e r n o f Clotrimazole.

i

8

JOHN G. HOOGERHEIDE AND BRUCE E. WYKA Table V X-Ray Powder D i f f r a c t m t e r n o f C l o t r i m e r o l e

28 9.172 9.398 9.817 10.203 12.361 14.178 15.159 15.641 16.645 l a . 517 15.415 19.830 20.647 22.502 22.983 24.101 24.200 24.266 25.086 25.519 26.177 27.474 28.132 28.354 28.810 30.923 30.999 31.318 31.461 31.783 32.655 34.182 34.503 34.577 36.980 37.080 37.196 37.374 37.598 37.680 37.778 37.057 37.989 37.998 38.513

d(!lr

9.641 9.410 9.009 8.669 7.161 6.247 5.345 5.665 5.326 4.791 4.572 4.477 4.302 3.951 3.870 3.693 3.678 3.668 3.550 3.490 3.404 3.246 3.172 3.148 3.099 2.892 2.885 2.856 2.843 2.815 2.742 2.623 2.599 2.594 2.431 2.424 2.417 2.406 2.392 2.387 2.381 2.376 2.372 2.368 2.337

a d ( i n t e r p l a n a r distance) = n A/2 s i n 1/11 = r e l a t i v e i n t e n s t t y

wb 80 10 47 10 100 19 7

7

32 a2 68 37 61 29 43 36 36 36 41 22 14 32 46 26 14 11 11 9 7 16 23 11 19 19 10 10 9 8 11 11 10 10 8 8 9

CLOTRIMAZOLE

2.8

241

Solubilitv

The s o l u b i l i t y o f c l o t r i m a z o l e has been d e t e r m i n e d i n solvents by using v i s u a l , g r a v i m e t r i c o r spectrophotom e t r i c analysis (Table V I ) .

2.9

D i s s o c i a t i o n Constant

The pKa o f c l o t r i m a z o l e i n 50% aqueous e t h a n o l i s r e p o r t e d t o be 4.7 ( 5 ) . Table V I S o l u b i l i t y o f C l o t r i m a z o l e i n Common S o l v e n t s Measured Solvent Acetone Benzene Chloroform Diethyl ether Dimet h y l formamide Dimet h y l s u l f o x i d e E t h a n o l USP Ethyl Acetate Met h ano 1 Mineral o i l Petroleum ether P o l y e t h y l e n e g l y c o l 400 P r o p y l ene g l yco 1 Water

(m

m

°

C

50

>loo >loo 14 >loo 45 95

45 >loo 0.8 1.1 60 35

<0.03

Met hod grav i m e t r i c visual visual g r av ime t r ic visual spec t r o p h o t omet r ic grav imetr i c g r av i m e t r i c visual spec t r o p h o t omet r ic spec t r o p h o t o m e t r ic spec t r o p h o t omet r ic spectrophotometr i c spectrophotometric

JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

242

3.

Synthesis

Clotrimazole i s synthesized by t h e r e a c t i o n o f o - c h l o r o t r i t y l c h l o r i d e w i t h i m i d a z o l e i n t h e presence o f a t e r t i a r y amine, as d e s c r i b e d b y Buechel, e t a l . ( 5 ) as shown i n F i g u r e 11. The y i e l d i n this-syythesis is solvent-dependent; reactions i n solvents w i t h h i g h d i e l e c t r i c constants g i v e the higher y i e l d s . Maul and S c h e r l i n g ( 9 ) used barium { 4C) c a r b o n a t e as s t a r t i n g m a t e r i a l t o s y n t h e s i z e ' ' C - c l o t r i m a z o l e . They made t h e i n t e r m e d i a t e s 2 - c t j l o r o - { c a r b o x y l C ) benzoic C 1 benzoylchloride, 2-chloroa c i d , 2-ch\ojo- { c a r b o x y l {carboxylC 1 benzophenone, ( 2 - c h l o r o p h e n y l d i p h e n y l { 1 4 C ) methanol and (2-chloropheny1)diphenyl - { ' C)methylchloride

en

route

tq

1-(2-~hlorophenyl)diphenyl-{

4.

1

radiolabelled clotrimazole, methyl-1H-imidazole. -

k1-

Stability

C l o t r i m a z o l e i s s t a b l e i n t h e s o l i d s t a t e unger normal s t o r a g e c o n d i t i o n s . It i s u n a f f e c t e d b y h e a t (70 C) and exposure t o d a y l i g h t f o r up t o two weeks (10). I n s o l u t i o n , t h e s t a b i l i t y o f c l o t r i m a z o l e i s pH dependent. I n an a l k a l i n e medium i t i s s t a b l e , b u t h y d r o l y z e s i n an a c i d i c medium t o ( 0 - c h l o r o p h e n y 1 ) diphenylmethanol p l u s i m i d a z o l e . Buechel e t a l . r e p o r t on, the relative hydrolytic stability of notrirnazole i n s o l u t i o n i n e t h a n o l - w a t e r and i s o p r o p a n o l - w a t e r m i x t u r e s under a c i d i c , n e u t r a l , and a l k a l i n e c o n d i t i o n s ( 4 ) . Thermal and 1 i g h t pH-stabi 1 i t y s t u d i e s have been performed. Known amounts o f c l o t r i m a z o l e were s e a l e d i n g l a s s ampuls a f t e r t h e a d d i t i o n o f 10 m l o f an aqueous b u f f e r . The pH range s t u d i e d was 1 t o 13. Table V I I g i v e s t h e r e s u l t s f o r samples s t o r e d a t 7 5 0 , 850, and 95OC f o r one week. R e s u l t s are g i v e n i n Table V I I I f o r samples s t o r e d i n t h e d a r k and u n d e r 350 f o o t - c a n d l e s o f f l u o r e s c e n t l i g h t f o r t h r e e months. Analyses were performed by u s i n g t h i n - l a y e r chromatoqraphy and e l u t i o n f o l l o w e d b y UV s p e c t r a l a n a l y s i s (10).

CLOTRIMAZOLE

243

Clp / PCI 3 CH2CI

UV-light

CI

CI

(1)

(I[)

cm 1

J (rn) F i g u r e 11

S y n t h e t i c Pathway t o C l o t r i m a z o l e I . 2-chlorobenzylchloride 11. 2-chlorobenzotrichloride I1 I . 2-chlorotri tyl chloride I V . Clotrirnazole Table V I I pH-Thermal S t a b i l i t y P r o f i l e Recovery o f C l o t r i r n a z o l e i n Percent*

pH Buffer -

State

1 2 4 4 6 7 8 10 13

solution part solution suspension suspension suspension suspension suspension suspension suspension

Hydroch 1o r i c a c i d C it r a t e Citrate Acetate Phosphate Phosphate Phosphate Borate Sodium h y d r o x i d e

* A f t e r one week.

75OC -

85OC --

95OC -

0 0 84 84 101 105 105 102 101

0 0 55 65 98 101 101 102 105

0 0 4 6 90 94 105 100 102

JOHN G . HOOGERHEIDE AND BRUCE E. WYKA

244

5.

Drug Metabolism and Pharmacokinetics 5.1

Drua Metabolism

After oral or topical administration clotrimazole undergoes r a p i d b i o t r a n s f o r m a t i o n i n t o i n a c t i v e met abolites. Duhm and c o - w o r k e r s (11) i s o l a t e d f i v e c l o t r i m a z o l e m e t a b o l i t e s f r o m r a t u r i n e and b i l e b u t found no c l o t r i m a z o l e . S t r u c t u r e s o f t h e metabol i t e s a r e shown i n F i g u r e 12. Human u r i n e and serum t e s t e d a f t e r o r a l doses o f c l o t r i m a z o l e c o n t a i n o n l y t r a c e amounts o f t h e d r u g substance. The two m a j o r m e t a b o l i t e s found i n u r i n e , serum, and b i l e a r e ( 2 - c h l o r o p h e n y l ) (4-hydroxypheny1)a phenylmethane and ( 2 - c h l o r o p h e n y l ) bis-phenylmethane; is s m a l l e r amount o f (2-chloropheny1)bis-phenylmethanol a1 so p r e s e n t . 5.2

Pharmacokinetics

E a r l y c l i n i c a l s t u d i e s employing o r a l administ r a t i o n o f c l o t r i m a z o l e p r o v i d e d d a t a on t h e pharmacok i n e t i c s o f s y s t e m i c a l l y d i s t r i b u t e d drug substance i n humans. As discussed above, metabolism of c l o t r i m a z o l e i s e x t r e m e l y r a p i d , and r e 1 i a b l e pharmacokinetics d a t a have been o b t a i n e d o n l y b y u s i n g a n a l y t i c a l m e t h o d s w h i c h In determine b o t h $tle d r u g substance and m e t a b o l i t e s . experiments w i t h C - l a b e l l e d drug, Duhm et a l . (11) found t h a t no c l o t r i m a z o l e was d e t e c t e d i n serum f o r a 20 m i n u t e i n t e r v a l a f t e r dosing. Peak serum l e v e l s o f up t o 4 u g h 1 were confirmed b y a number o f workers (1,12-15); these l e v e l s were reached between two and f o u r h o u r s a f t e r d o s i n g i n a d u l t s and a t about s i x hours a f t e r a d m i n i s t r a t i o n t o children. Rosenkrantz and P u e t t e r (16) e s t i m a t e d t h a t up t o 98% o f c l o t r i m a z o l e i n serum i s bound t o serum p r o t e i n s . Several workers have f o l l o w e d systemic c l o t r imazole c l e a r a n c e b y d e t e r m i n i n g t h e d r u g and m e t a b o l i t e s e x c r e t e d i n urine. W e i n g a e r t n e r , e t a l . ( 1 7 ) f o u n d no d r u g substance i n u r i n e u n t i l b e t w e e n 3 0 and 60 minutes a f t e r a d m i n i s t r a t i o n ; t h e y r e p o r t e d peak u r i n e c o n c e n t r a t i o n s a t between 6 and 1 2 h o u r s . Plempel and co-workers (1) have r e p o r t e d peak u r i n e c o n c e n t r a t i o n s o f 30 t o 60 u g h 1 1 2 t o 14 hours a f t e r d o s i n g .

CLOTRIMAZOLE

245

Table V I I I pH-L i g h t S t a b i l i t y P r o f i l e Recovery o f C l o t r i m a z o l e i n Percent** Light Dark -

pH Buffer -

State

1

sol ut ion part solution suspension suspension suspension suspension suspension suspension

2 4 6 7 8 10 13

Hydrochloric acid C it r a t e Citrate Phosphate Phosphate Phosphate Borate Sod iurn h y d r o x i d e

9 20 98 97 99 101 99 103

12 41 102 103 102 103 103 99

**Stored i n t h e dark and under 350 f o o t - c a n d l e s o f f l u o r e s c e n t 1 i g h t for 3 months.

I

OH

I

OH

CI

F i g u r e 12. C l o t r i m a z o l e M e t a b o l i t e s : I . (2-chlorophenyl )diphenylmethanol I I . ( 2-chl orophenyl ) d i phenylmethane

III. IV. V.

(2-chlorophenyl ) , (4-hydroxyphenyl ) phenylmethane (2-chlorophenyl ) , (4-hydroxyphenyl ) p h e n y l me t h a no1 2-chlorobenzophenone

JOHN G. HOOGERHEIDE AND BRUCE E. WYKA

246

Pharmacokinetics o f t o p i c a l l y appl i e d c l o t r i m a z o l e has been s t u d i e d i n s e v e r a l l a b o r a t o r i e s . Duhm a n d co-workers ( 1 8 ) found t h a t w h i l e C - c l o t r i m a z o l e appl i e d as a cream p e n e t r a t e d t h e s k i n t o a d e p t h o f 2000 bm, no d r u g s u b s t a n c e o r m e t a b o l i t e s were d e t e c t e d i n t h e serum. A s m a l l q u a n t i t y o f t h e m a t e r i a l ( u p t o 0.4%) was d e t e c t e d i n t h e u r i n e over a f i v e - d a y p e r i o d . In a s i m i l a r study, H o l t ( 1 9 ) used a m i c r o b i o l o g i c a l a s s a y w i t h a d e t e c t i o n l i m i t o f 0.01 pg/ml; over a t h i r t y - d a y p e r i o d o f c l o t r i m a z o l e c r e a m a p p l i c a t i o n h e o b s e r v e d no d r u g et al. (20) s u b s t a n c e i n e i t h e r serum o r u r i n e . Wallace, n o t e d t h a t 24 h o u r s a f t e r a t e n - d a y t r e a t m e n t w i t h clotrimazole t o p i c a l preparations, drug l e v e l s i n t h e skin were as h i g h as 2 pg/mg; t h i s v a l u e d e c r e a s e d t o a b o u t 0.4 vg/mg a f t e r f o u r d a y s .

In studies using c l o t r i m a z o l e vaginal t a b l e t s , 100 mg, Duhm e t a l . ( 1 8 ) f o u n d peak serum l e v e l s o f 0.03 pg/ml a f t e r 2 4 h o u r s . P h a r m a c o k i n e t i c s and p h a r m a c o l o g y o f c l o t r i m a z o l e a r e f u r t h e r c o v e r e d i n a c o m p r e h e n s i v e summary b y Sawyer, e t a1 ( 2 1 ) as we1 1 as i n r e v i e w s b y Meade ( 2 2 ) and Seneca

.

v3r 6.

Methods o f A n a l y s i s 6.1

Elemental A n a l y s i s

Conventional procedures f o r t h e d e t e r m i n a t i o n o f C,H,N, and C1 y i e l d e d t h e f o l l o w i n g r e s u l t s f o r a sample c o n f o r m i n g t o USP XX s p e c i f i c a t i o n s .

E 1ement C H N c1

Found 76.63 4.93 8.12 10.18

% -

Theory 76.63 4.97 8.12 10.28

CLOTRIMAZOLE

6.2

247

Identification

S e v e r a l methods have been proposed f o r t h e identification o f clotrimazole. Kuhnert-Brandstaetter, e t a l . ( 8 ) observed t h a t t h e d r u g s u b s t a n c e f o r m s e u t g c t i c s w i t h p h e n a c e t i n and b e n z a n i l i d e w h i c h m e l t a t 110 and 115 C, r e s p e c t i v e l y . D a t a on e u t e c t i c m e l t i n g p o i n t s i s to c o n f i r m t h e i d e n t i t y o f c l o t r i m a z o l e , w h i c h m e l t s used o a t 143 C . K r i h a r , e t a l . ( 2 4 ) have c h a r a c t e r i z e d t h e u l t r a v i o l e t s p e c t r a o f T o t r i m a z o l e i n m e t h a n o l and i n 0.1N HC1 as an a i d t o t h e i d e n t i f i c a t i o n o f t h i s d r u g substance, The USP X X i d e n t i f i c a t i o n t e s t s f o r c l o t r i m a z o l e ( 2 5 ) i n c l u d e b o t h t h i n - l a y e r chromatography, i n w h i c h t h e sample s p o t must appear a t t h e same R f v a l u e as r e f e r e n c e s t a n d a r d m a t e r i a l , and i n f r a r e d s p e c t r o p h o t o m e t r y . 6.3

Spectrophotometric Analysis

L i m i t e d u s e h a s b e e n made o f u l t r a v i o l e t s p e c t r o p h o t o m e t r y i n t h e a n a l y s i s o f c l o t r i m a z o l e due t o i t s low a b s o r p t i v i t y . Kr6&nar, e t a l . ( 2 4 ) suggested t h a t such a n a l y s e s s h o u l d be p o s s i b l e ; S z a b o l c s ( 2 6 ) used m e a s u r e m e n t s a t 2 6 1 nm t o a s s a y c l o t r i m a z o l e i n formulations. Upon h e a t i n g w i t h t r i c h l o r o a c e t i c o r p e r c h l o r i c a c i d s , s o l u t i o n s o f c l o t r i m a z o l e become b r i g h t y e l l o w ; t h e c o l o r fades r a p i d l y w i t h t r i c h l o r o a c e t i c a c i d b u t p e r s i s t s with perchloric acid (1). T h i s r e a c t i o n , which forms t h e b a s i s f o r a c o l o r i m e t r i c assay o f t h e d r u g s u b s t a n c e , g i v e s s o l u t i o n s which obey B e e r ' s l a w up t o c o n c e n t r a t i o n s o f 1 0 pg/ml when r e a d a t 436 nm ( 2 7 ) . T h i s method has been used f o r t h e e s t i m a t i o n o f c l o t r i m a z o l e i n b i o l o g i c a l f l u i d s and t i s s u e s a f t e r e i t h e r s o l v e n t e x t r a c t i o n (16,27) o r extraction followed by thin-layer chromatography (1,13,17,28).

JOHN G.HOOGERHEIDE AND BRUCE E. WYKA

248

6.4

T i tr i m e t r i c An a1y s is

Assay o f c l o t r i m a z o l e b u l k d r u g s u b s t a n c e i s p e r f o r m e d b y nonaqueous t i t r a t i o n ( 2 5 ) . The s a m p l e d i s s o l v e d i n g l a c i a l a c e t i c a c i d i s t i t r a t e d w i t h 0.1M perchloric acid i n glacial acetic acid t o a green e n d - p o i n t ; p - n a p h t h o l b e n z e i n i s used as i n d i c a t o r . Each m i l l i l i t e r o f 0.1M p e r c h l o r i c a c i d i s e q u i v a l e n t t o 34.48 m i l 1i g r a m s o f c l o t T i m a z o l e . A f t e r e x t r a c t i o n with c h l o r o f o r m , Szabolcs ( 2 6 ) assayed c l o t r i m a z o l e i n p h a r m a c e u t i c a l f o r m u l a t i o n s b y t i t r a t i o n w i t h 0.1M - p e r c h l o r i c a c i d ; g e n t i a n v i o l e t was used as i n d i c a t o r .

A two-phase t i t r a t i o n method developed b y P e l l e r i n , e t a l . ( 2 9 - 3 1 ) s e r v e s as a b a s i s f o r t h e t i t r a t i o n o f c T o t r i m a z o l e w i t h sodium l a u r y l s u l f a t e . Samples o f c l o t r i m a z o l e f o r m u l a t i o n s a r e p a r t i t i o n e d between c h l o r o f o r m and 2N s u l f u r i c a c i d . M e t h y l y e l l o w i s a d d e d as i n d i c a t o r a n d t h e m i x t u r e t i t r a t e d w i t h s t a n d a r d i z e d sodium l a u r y l s u l f a t e i n w a t e r . The end p o i n t i s r e a c h e d when t h e c h l o r o f o r m l a y e r t u r n s g o l d - o r a n g e ( 3 2 ) . T h i s s t a b i l i t y - i n d i c a t i n g t i t r a t i o n has been used t o assay c l o t r i m a z o l e i n f o r m u l a t i o n s (32,33) and t o f o l l o w h y d r o l y s i s o f t h e drug substance ( 5 ) . 6.5

Chromatograph ic A n a l y s i s 6.5.1

Paper Chromatography

C l o t r i m a z o l e c a n be s e p a r a t e d f r o m i t s i m p u r i t i e s and d e g r a d a t i o n p r o d u c t s b y d e s c e n d i n g p a p e r c h r o m a t o g r a p h y (10). The method uses paper i m p r e g n a t e d w i t h p r o p y l e n e g l y c o l and a m o b i l e phase o f p r o p y l e n e g l y c o l - s a t u r a t e d l i g r o i n . C l o t r i m a z o l e i s d e t e c t e d a t R f = 0.4 b y s p r a y i n g w i t h Dragendorff reagent, 6.5.2

Th i n - L a y e r Chromatography

T h i n - l a y e r c h r o m a t o g r a p h y (TLC) o n s i l i c a g e l h a s been used e x t e n s i v e l y t o s e p a r a t e c l o t r i m a z o l e f r o m f o r m u l a t i o n and b i o l o g i c a l m a t r i c e s p r i o r t o q u a n t i t a t i o n . A summary o f t h i n - l a y e r a d s o r b e n t s and m o b i l e phases used i n these separations i s g i v e n i n Table I X .

Table I X Th in-L ayer Chromatography Systems f o r C 1o t r imazol e So 1vent (see below)

P l a t e Medium ( see be1ow)

1 s t Dimension : 2nd Dimens i o n :

Detect i o n (see below)

I

R f Value

Reference

0.57 0.64

9

1 s t Development: 2nd Development:

I1

-

17

1 s t Development: 2nd Development:

I1

-

13

1 s t Development: 2nd Development:

TI1

0.4

27

F

I11

0.55

27

C

I1

0.3

1,28

G

IV

0.65

25

H

IV,V

0.4

25

J

IV,V

0.85

10

JOHN G. HOOGERHEIDEAND BRUCE E. W Y K A

250

Table IX (continued) Plate Medium a. Silica gel 60.

b. Silica gel foil (Polygram Sil NH.R, MacheryNagel ) . c.

Silica gel 60 F .

Solvent System

A. Benzene:methanol (4:l). B. Methanol. C. Chloroform.

D. Petroleum ether (40-60°):acetone: benzene:ethanol :pyr id ine (70:12:10:7:1).

E. Benzene. F. Petroleum ether (40-60') :ethylacetate:acetone:ethanol : ammon i a ( 25%) (45:25 :25 : 5 :0.5 ) .

G. Xy1ene:n-propanol :ammonia (180:20:1).

H. Ether equilibrated with ammonia vapor.

J. Ethyl acetate:ammonia (25%) (98:2). Detection Method

I. Autoradiography. 11. 111. IV.

V.

Trichloroacetic acid in n-butylacetate. Spray sequentially with ethanolic iodine, sodium carbonate, and sulfanil ic acid/sodium nitrite. Fluorescence quenching under short-wave ultraviolet light. Spray with Dragendorff reagent.

25 1

CLOTRIMAZOLE

Q u a n t i t a t i v e a n a l y s e s e m p l o y i n g TLC h a v e b e e n c a r r i e d o u t i n s e v e r a l ways. R i t t e r , e t a l . (27) used densitometry t o determine c l o t r i m a z o l e t h e TLC p l a t e a f t e r formation o f a red s u l f a n i l i c acid derivative. Their c a l i b r a t i o n c u r v e s were l i n e a r between 0.5 and 4 p g c l o t r i mazole per spot. O t h e r i n v e s t i g a t o r s have d e t e r m i n e d t h e d r u g substance s p e c t r o p h o t o m e t r i c a l l y b y s c r a p i n g t h e bands o f f t h e TLC p l a t e and r e a c t i n g e i t h e r w i t h b u t y l a c e t a t e and p e r c h l o r i c a c i d (1,13,17,28) o r w i t h bromphenol b l u e

(10). 6.5.3

Gas Chromatography

C l o t r i m a z o l e i n human s k i n s a m p l e s h a s b e e n d e t e r m i n e d b y gas c h r o m a t o g r a p h y w i t h e l e c t r o n - c a p t u r e detection (20,34). Samples were e x t r a c t e d w i t h e t h e r , d r i e d , r e d i s s o l v e d i n benzene and c h r o m a t o g r a p h e d on 6', 1/8" g l a s s columns packed wdth 3% OV-17 on Gas Chrom Q. Column t e m p e r a t u r e was 250 C and t h e c a r r i e r gas was S t a n d a r d c u r v e s were l i n e a r argon-methane a t 9 ml/min. o v e r t h e r a n g e o f 1-25 ng i n j e c t e d .

6.5.4

H i g h P e r f o r m a n c e L i q u i d Chromatography

S t a b i l i t y - i n d i c a t i n g assays o f c l o t r i m a z o l e i n t h e b u l k d r u g s u b s t a n c e and i n f o r m u l a t i o n s have been c a r r i e d o u t b y h i g h performance 1 i q u i d c h r o m a t o g r a p h y ( 3 5 ) . The a n a l y s e s were p e r f o r m e d on a IA Bondapak C i a column w i t h a m o b i l e phase o f methanol:O.O25M K 2 H P h ( 3 : l ) a t 1 . 0 m l / m i n . Chromatographic r e s p o n s e was l i n e a r f r o m 2 t o 40 p g c l o t r i mazole i n j e c t e d . Average r e c o v e r y o f d r u g s u b s t a n c e f r o m f o r m u l a t e d m a t e r i a l s r a n g e d f r o m 99.5 t o 100.0 p e r c e n t . A n a l y s e s were r e p r o d u c i b l e , w i t h between-day r e l a t i v e s t a n d a r d d e v i a t i o n s between 0.6 and 1.8 p e r c e n t .

6.6

Radiochemical A n a l y s i s 14

Duhm, e t a l . (18,36) used C-labelled c l o t r i mazole t o detEmTne drug d i s t r i b u t i o n s i n b i o l o g i c a l samples. R a d i o a c t i v i t y was d e t e r m i n e d i n serum and u r i n e b y t h e use o f l i q u i d s c i n t i l l a t o r s c o n t a i n i n g 8 g / 1 o f b u t y l PBD i n t o l u e n e : d i o x a n e * e t h a n o l ( 1 : l : l ) . S k i n samples were burned; t h e r e s u l t i n g I4C02 was absorbed i n base and c o u n t e d b y means o f s c i n t i11 a t o r s .

252

JOHN G. HOOGERHEIDE AND BRUCE E. WYKA

6.7

Microcalorimetr i c Analysis

I n h i b i t i o n o f t h e r e s p i r a t i o n o f Saccharomyces c e r e v i s i a e b y a n t i f u n g a l agents p r o v i d e s t h e b a s i s f o r a s e n s i t i v e c l o t r i m a z o l e assay. Beezer and c o - w o r k e r s ( 3 7 ) m o n i t o r e d r e s p i r a t i o n m i c r o c a l o r i m e t r i c a l l y b e f o r e and a f t e r a d d i t i o n o f c l o t r i m a z o l e ; degree o f i n h i b i t i o n o f r e s p i r a t i o n was r e l a t e d t o t h e c o n c e n t r a t i o n of c l o t r i m a z o l e . The minimum d r u g c o n c e n t r a t i o n d e t e c t a b l e b y t h i s method was 3 x 10’5M.6.8

M i c r o b io 1og i c a l A n a l y s i s

M i c r o b i o l o g i c a l assays o f c l o t r i m a z o l e a r e a g a r d i f f u s i o n assays based on a c o m p a r i s o n between t h e g r o w t h i n h i b i t i o n zones p r o d u c e d b y s t a n d a r d s o l u t i o n s and t h o s e produced b y t e s t samples. The t e s t o r g a n i s m C a n d i d a p s e u d o t r o i c a l i s v a r . c a r s h a l t o n has been used b y several *12,27,38) t o a s s a y c l o t r i m a z o l e i n serum, u r i n e , and f e c e s . When t e s t i n g t h e s t a b i l i t y o f c l o t r i m a z o l e i n c u l t u r e medium, H o e p r i c h and Huston ( 3 9 ) employed K l u v e r o m c e s f r a i l i s as t e s t o r g a n i s m . In a s t u d y o f c o t r i m a z o l e s t a i i t v on samDle d i s c s . S a u b o l l e and i o e p r i c h (40) used Candida” a l b i c a n s as t e s t ’ o r g a n i s m . H o l t ( 4 1 ) has d i s c u s s e d s e o f s e v e r a l o r g a n i s m s and assay t y p e s i n t h e a n a l y s i s o f a n t i f u n g a l d r u g s , i n c l u d i n g c l o t r i m a z o l e.

++

Two p a p e r s h a v e compared m i c r o b i o l o g i c a l a s s a y r e s u l t s t o t h i n - l a y e r chromatographic determinations o f c l o t r i m a z o l e (27,42). 7.

Acknowledaements

The a u t h o r s w i s h t o t h a n k t h e S c h e r i n g C o r p o r a t i o n Research L i b r a r y and P h y s i c a l and A n a l y t i c a l C h e m i s t r y S t a f f s , i n p a r t i c u l a r Ms. Jean Nocka, Ms. M i c a e l a K a t z , D r . Henry S u r p r e n a n t , D r . Mohindar S. Puar, Ms. Jane L i m p e r t , and Ms. L a u r e l Andersen, f o r t h e i r a s s i s t a n c e i n the preparation o f t h i s analytical p r o f i l e .

CLOTRIMAZOLE

253

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Talanta

847 ( 1 9 6 5 ) .

32.

U n i t e d S t a t e s Pharmacopeia XX, p . 160.

33.

Kudo, A.,

34.

Wallace, S. M., Shah, V. P., Riegelman, S., E p s t e i n , W. L., Anal. Lett. 611, 461 (1978).

35.

Hoogerheide, J. G . , S t r u s i a k , S. H., Taddei, C. R., Townley, E. R., and Wyka, B. E., J. Assoc.O f f. Anal. Chem. ( t o be p u b l i s h e d J u l y , 1 9 8 l r .

36.

Medenwald, H., P a t z s c h k e , K., Duhm, B., Maul, W., Wegner, L . A. and P u e t t e r , J., P o s t g r a d . Med. J. 50, 13 (1974), Suppl 1.

Jap. -J. -C l i n . Rep. 6,

85 (1972). and

.

37.

Beezer, A., Chowdhry, 6. Z., N e w e l l , R. D., T y r r e l l , H. J . V., A n a l. Chem. 49, 1781 ( 1 9 7 7 ) . -

38.

H o l t , R. J. and Neman, R . L., J. C l i n . P a t h o l . 25,

and

1089 (1972). 39.

Hoeprich,

C., A b s t r a c t 104, Antimicrob A X s San F r a n c i s c o , CA, S e p t . 11-13 (i97*

P.

D.

and Huston,

A.

Proc. 1 4 t h I n t e r s c i e n c e Conf. Chemother.,

40.

S a u b o l l e , M. A. and H o e p r i c h , P. D., A b s t r a c t 102, Proc. 14th I n t e r s c i e n c e Conf. A n t i m i c r o b . A q X s Chemother., San F r a n c i s c o , CA, S e p t . 11-13 ( 1 9 7 4 ) .

41.

H o l t , R. J . , J . C l i n . P a t h o l . 28, 767 ( 1 9 7 5 ) .

42.

H o l t , R.J., Abs. P r o c . 1 0 t h I n t e r n a t . M i c r o b i o l , Mexico, p . m , A C 9 - l m .

.

L i t e r a t u r e s u r v e y t e r m i n a t e d December, 1980.

Cong.

DOPAMINE HYDROCHLORIDE James E . Carter, John H . Johnson, and David M. Bamke

1.

2.

3. 4.

5. 6. 7. 8.

Description 1.1 Chemical and Proprietary Names 1.2 Empirical Formula 1.3 Appearance, Color, and Odor Physical Properties 2.1 Melting Range 2.2 Solubility Profile 2.3 Infrared Spectrum 2.4 Ultraviolet Spectrum 2.5 Proton Magnetic Resonance Spectrum 2.6 "C Magnetic Resonance Spectrum 2.7 Mass Spectra 2.8 Differential Scanning Calorimetry 2.9 Crystal Properties Synthesis Analysis 4.1 Elemental Analysis 4.2 Colorimetric Assay 4.3 Nonaqueous Titration 4.4 Thin-Layer Chromatography (TLC) 4.5 Gas Chromatography 4.6 High Performance Liquid Chromatography (HPLC) Stability Analysis of Biological Samples Metabolism and Excretion Acknowledgement References

Analytical Profiles of Drug Substances Volume I I

257

258 258 258 258 258 258 259 259 259 259 263 265 266 266 268 269 269 269 269 269 270 270 270 27 1 27 1 27 1 272

Copyright 0 1982 by The American Pharmaceutical Association ISBN 012-260811-9

JAMES E. CARTER E T A L .

258

1.

Description 1.1 chemical and Proprietary Names

Dopamine hydrochloride is the mn-proprietary name for 4-(2-aminoethyl)-1,2-benzenediol hydrochloride. The free base (dopamine) has also been known in the chemical literature as 3-hydroxytyramine and 3,4 dihydroxyphen~thylamine. The drug is available generically for the correction of hamdynamic imbalances present in the shock syndmm due to myocardial infarction, t r a m , endotoxic septicemia, open heart surgery, renal failure and chronic cardiac decanpensation as in congestive heart failure (1). Proprietary names include Cardiosteril, Docard, Dopamine Fabre, Dopamine Gullini, Dopastat, Dynatra, Intropin, Intropinject, Intropine and Orion. 1.2 Empirical F o d a C8H12Cm2 Wlecular Weight 189.64 Structure

1.3 Appearance, Color

&

Odor

Dopamine hydrochloride is a white to off white odorless crystalline pcxder.

2.

Physical Properties 2.1 Melting range

The Merck Index reports the mlting point of the hydrochloride salt as 241OC with decanposition (2). The

259

DOPAMINE HYDROCHLORIDE

equilibrium melting range ( i n the absence of a i r ) has been established a s 245OC - 246OC with deccenposition ( 3 ) . 2.2

Solubility Profile

Dopamine hydrochloride is f r e e l y soluble i n water: soluble i n mthanol and hot ethanol: p r a c t i c a l l y insoluble in ether, chloroform, benzene and toluene. Dopamine hydrochloride is soluble in aqueous solutions of a l k a l i hydroxides. 2.3

Infrared Spectrum

The KBr pellet infrared spectrum of 0.5% d o w e hydrochloride obtained with a Perkin-Elmer 283 Infrared Spectrophotomter is contained i n Figgie 1. The b r q d strong absorption bands appearing a t 3350 cm and 3230 cm are due to the O-H and N-H stretching vibrations. The i n t e m l e c u l a r and i n t r a m l e c u l a r hydrogen bonding s h i f t s these absorption bands to-lower frequencies than a f r e e h y d r o q l group (3700 3500 an ) and a f r e e a q n e (3500 - 3300 an ) The -1 aromatic (3100 - 3000 cm ) and a l i p h a t i c (3000 2900 cm ) C-H stretching bands a r e prevent as expected. The N-H bending vibrations 25 tile NH group shaw medium absorption centered a t 1610 cm The 2-H out of plane bending of the 1 , 2 , 4 t r i s u b s t i t u t e d F e n e ring i indicated by -B the sharp bands a t 876 cm and 812 cm

.

-

.

.

2.4

Ul'zaviolet

spectrum

Dopamine hydrochloride exhibits a single absorption peak centered a t 280 nm w i t h a mlar absorptivity (E) of 2707 (Figure 2 ) . The spectrum was obtained with a Beclanan Acta I11 double beam spectro@otometer. 2.5

Proton Maanetic Resonance S m t r u m

The 60 MHz proton magnetic resonance spectrum was obtained with a Varian Associates T-60A spectrcneter. The spectrum i n CD OD with tetramethylsilane (TMS) as internal reference is d n t a h e d i n Figure 3. The s p l i t i n g patterns are not simple. Howsver, t h e integration and qeneral locations of psaks are consistent w i t h the structure. The peaks a t 0.8 and 3.35 are due to mall amounts of CH30H in the deuterated solvent.

Fig. 1

KBr Infrared Spectrum of Dopamjne Hydrochloride. Instmnent: Perkin-Elmer MAel 283

Oopamfne H y d r o c h l o r i d e UV Spectrum

Scan Speed Chart Expansion C o n c e n t r r t l o n Span

0 . 5 nmlscc 20 nmlinch 1.0 deuterium 280 nm 2.109 x 10-4 n o l e / L 0.571 2707 1% Sodium B i r u l f i t e

Concentration Absorbance Nolar AbsorDtivlty Reference Cell U.UrekSv;& Operator Date t i - i 2 - # /

I

240

Fig. 2 Instrument:

240

I

SbO

200

$0

nm

Ultraviolet spectrum of IXpamhe Hydrochloride BeclaMn Acta 111 261

I

r

:

I

I

I

I!

I

I

f---

li

I I I I 1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . . I . . . . I . . . . I . 1 . . . I 80

Fig. 3

7 0

60

50

PPM:d:

4 0

30

20

Proton Magnetic Resonance Spectnnn of Dopamine Hydrochloride in CD3OD Instrument: Varian T-60A

10

I 0

263

DOPAMINE HYDROCHLORIDE

Chemical s h i f t s (b) i n ppn r e l a t i v e to TMS are:

b

H

Proton Assigment a b C

2.6

O

G

C

# of Protons 4 5 3

H ,-CH ,-NH ,.HCI

Chemical S h i f t (b) 3.0 4.95 6.75

Mu1t i p l i c i t y

multiplet multiplet multiplet

I3C Magnetic Resoname Spectrum

The "C analysis of dopamine hydrochloride was taken on a JEOL FX-270 superconductiny NMR operating a t a frequency of 67.83 IWz using a 45" (9 usec) pulse, a 3.6 second r e p e t i t i o n rate and 16384 data points ( 4 ) . The sample was dissolved i n D20; P-pfoxane was added. as a reference. Spectra of the e n t i r e C range were obtained with ccmplete decoupling and gated decoupling with Nuclear Overhauser Effect (NOE) to obtain the chemical s h i f t s and coupling constants. The ccmpletely decoupled sFectrum is sham i n F j w e 4. The resolution of a l l a r m t i c carbons (not cham in Figure 4 ) was obtained using a 2.5 kHz width. The assignrrent of t h e a r m t i c carbon l i n e s was aided by canparison to 3,4 dihydroxybenzene.

Fig. 4

l 3 C Magnetic Resonance Spectnnn of Dopamine Hydrochloride w i t h p-dioxane as reference. Instrurtlent: JExlL FX 270

265

DOPAMINE HYDROCHLORIDE

With the carbons n-red as indicated below the chemical shifts and coupling constants are as s h m in T a b l e I.

r 2

HO

Table I. I3C assignments and C-H

coupling constants for

Dopamine Hydrochloride

Carbon Number

Shift (ppn) 129.1 116.3 143.9 142.7 116.4 121.1 32.0 4c.7

Jm(Hz)

159.3

-

156.7 160.0 128.3 143.9

2.7 Mass Spectra The electron inpact (EI) mass spectnrm at 70 eV and the methane derived chemical ionization (CI) mass spectrum were obtained with a Hewlett-Packard 5985 quadraple mass spectrcaneter (5).

The CI spectrum contains t w prcaninent ions at n4e 137.0 and m/e 154.0. The m/e 154.0 corresponds to C H NO which is protonated dopamine. Loss of m n i a leave8 which is the base peak i n the spec=tnrm at m/e 137.0.

&gg8;

JAMES E. CARTER ETAL.

266

The E I spectrum i s surprisingly simple. The base peak a t m/e 124.1 corresponds to C H 0 The intact dopamine a t m/e 153.0 i s seen in &e8s&ctrum. The m/e 77.0 and m/e 78.l+are due $0 the a m t i c r i n g and correspond t o C H and C F respectively. The hydrochloride s812 is no$ geen i n e i t h e r the E I o r C I spectrum which js as expected. 2.8

Dif ferenti a1 Scanning Calorink=tq

Dopamine hydrochloride was heated a t a rate of 10' per minute i n a Perkin-Elmr DSC-2 d i f f e r e n t i a l scanning A single sharp endothem calorimeter frcan 460'K t o 530'K. w a s observed with an onset temperature of 516'K (243OC) and with the endothem maximum a t 519.8'K (246.8'C). An accurate heat of t r a n s i t i o n bH) could m t be calculated because of deccanposition upon rwlting. 2.9

C r y s t a l Properties

Crystals f r m a representative l o t of dopamine hydrochloride are predaninantly triangular p l a t e s ( 3 ) . The o p t i c a l crystallographic examination indicated t h a t the crystals are triclinic or Fossibly m n o c l i n i c . X-ray powder patterns w e r e obtained w i t h copper radiation and a nickel f i l t e r . The d-spacings and r e l a t i v e k t e n s i t i e s (I/I ) are listed i n T a b l e 11. The f i r s t column shws the p a t t e r 8 f o r the drug a f t e r grinding to break up t h e p l a t e s and therefore the preferred o r i e n t a t i o n of the c r y s t a l s . Column 2 shows the d i f f r a c t i o n pattern of the crystals without p r i o r grinding. The absence of several key d i f f r a c t i o n l i n e s and the reduced influence of o t h e r s i n this orientation is c l e a r l y d m n s t r a t e d by canparing t h e two columns.

DOPAMINE HYDROCHLORIDE

267

TABLE I1 X-ray Diffraction M e r Patterns of Dopamine Hydrochloride

Ground Sample d-spcing I/Io

6.37 5.56 5.50 5.24 4.57 4.18 4.07 3.98 3.81 3.72 3.53 3.49 3.43 3.23 3.19 2.96 2.83 2.77 2.75 2.69 2.63 2.62 2.55 2.41 2.27 2.17 1.90

5 30 60 5 75 95 30 100 65 60 70 45 70 65 45 10 10 55 45 10 60 60 60 10 10 20 10

1.86

10

Unground Sample d-spacing IDo

5.56

10

5.24 4.58 4.19 4.07 3.97 3.82 3.71 3.53 3.49 3.43 3.23

20 15 30 15 15 100 15 30 65 20 20

2.97 2.83 2.77

5 5 15

2.62

80

2.55

15

2.17 2.09 1.99 1.96 1.92 1.90

10 5 5 5 5 5

JAMES E. CARTER E T A L . 3.

Synthesis

Dopamine hydrochloride i s available from a variety of ccprmercial sources. Details of the synthetic process are considered proprietary information. However, one workab1.e process using known reactions and readily available materials is shown in Figure 5 ( 6 ) .

cH30D CH=CHNOp H2

CH30~cHoCH3N02

CH,O

CH3O

cH30x3 CH2CH2NH2

H Br

CH 3O

HO

Fig. 5 Synthetic Scheme for Dopamine Hydrochloride

The d h t h o q p h e n e t h v l a r h e (knma s homveratrylamine) has been converted to dopamine hydrochloride in one step with w i d b e hydrochloride ( 7 ) .

269

DOPAMINE HYDROCHLORIDE

4.

Analysis 4.1

E l m t a l Analysis

Elerwntal analysis of a typical dopamine hydrochloride sample is as follaws: Element C H

c1 N 0

4.2

% Theoretical

50.67 6.38 18.70 7.39 16.87

% Found

50.51 6.55 I -

7.37

---

Colorimetric Assay

A colorimetric assay f o r the analysis of d0pami.m has been adapted f m The United S t a t e s Phaxmampeia Epinephrine Assay ( 8 ) . "The Photarnetric Detection of

Adrenaline in P h a r m e u t i c a l Products" was f i r s t described by Jhty in 1948 ( 9 ) . 4.3

Non-aqueous T i t r a t i o n

The purity of dopamine hydrochloride may be assessed by non-aqueous t i t r a t i o n with crystal violet as the indicator. The hydrochloride salt is dissolved in g l a c i a l acetic acid, mercuric acetate is added to remove the chloride as unionized mercuric chloride, crystal violet is added and the amine is titrated to a green end-point with 0.1 N p r c h l o r i c acid.

-

4.4

Thin-layer Chromatoqraphy (TLC)

Thin-layer chrormtography on silica g e l is p a r t i c u l a r l y helpful in assessing the purity of the r a w drug. I n a solvent system of ethyl acetate-methanolm n i n u n hydrGxide (85:10 :5) a l l p o t e n t i a l wthoxy-phenethylamine impurities are readily separated and differentiated. The 3-hydroxy-4-methoxyphenethylamine and dopamine fluoresce under s h o r t wave ultraviolet l i g h t . After spraying w i t h ninhydrin and developing a t 110' f o r 5 minutes dopamine appears as a brown spot (R N 0.07) , the f 3-hYdr0~-4-~thO analog ~ appears as a yellaw spot (Rf N 0 . 2 2 ) , the 3-methoxy-4-hydroxy analog appears as a pink spot (Rf N 0.26) and the dimethoxy compound a p p a r s as a pink spot (Rf N 0.30).

JAMES E. CARTER ETAL.

270

I n a solvent system of n-buitanol-glacial acetic acid-water (12:3:5), doparnine has an R of approximately 0.6. f The canpounds may also be visualized by spraying t h e silica p l a t e with 0.5% iodine i n chloroform. 4.5

Gas ChmnatouraDhv

Because of the high-boilirLg, polar and oxidizable nature of dopamine, it i s not readily amenable to gas chrmtography. 4.6

High Performance Liquid Chrmtography (HFTC)

N o work has been reported for t h e HPLC analysis of dopamine hydroFhloride i n pharmaceutical dosage forms as might be expected f o r a non-patented product. One relevant papr reports t h e chrmatographic behavior of dopamine and related catecholamines i n a reverse phase system on t h e ubiquitous octadecylsilane column (10) The authors found dopamine to be insensitive t o pH changes b e b e e n 2 and 5 w i t h n i t r i c acid but to be retained longer as t h e pH increased in acetic acid. Doparnine appeared to form ion pairs being retained f o r longer times with t r i c h l o r o a c e t i c acid and octylsulfonic acid than with mineral acids.

.

5.

Stability

Dopamine hydrochloride ( l i k e other catecholamines) i s r e l a t i v e l y unstable t o heat, l i g h t and oxygen. The presence of oxidizing agents or trace mtals such as copper or iron also increases t h e rate of degradation. Properly stored i n g l a s s containers t h e raw drug i s stable f o r three to f i v e years.

Dopamine hydrochloride is generally f o m l a t e d with 1% sodium b i s u l f i t e as an antioxidant. The r e s u l t a n t solution has a pH of about 4 and when properly protected f r m a i r , heat and l i g h t i s s t a b l e f o r three to f i v e years. The s t a b i l i t y and compatibility of dopamine solutions with c m n intravenous f l u i d s (11)various antibioiics (12) and miscellaneous drugs and additives w i t h which it could be potentially mixed (13) has been reported. Dopamine i s unstable a t an aklaline pH. Thus dopamine solutions are not stable i n sodium bicarbonate but are stable i n dextrose, sodium chloride, sodium lactate, l a c t a t e d Ringer's solution, and mannitol.

DOPAMINE HYDROCHLORIDE

6.

27 1

Analysis of Biological Samples

Because of its rapid metabolism, methods f o r the routine analysis of dopamine i n blood and urine have not been developed. Methods which have been reported are generally concerned with endogenous dopamine and its mtabolism rather than the clinical pharmacology and analysis of infused dopamine. The analysis of l 4 C - d o m e and its radiolabelled metabolities following an infusion i n human subjects has been reported ( 1 4 ) . A novel approach which may be applicable to analysis following a dopamine infusion has recently been developed (15). This method converts dopamine (and other catechols) t o i t s mthoxy derivative i n the presence of catechol-cmethyltransferase and S-adenosylmthionine- H methyl. The t r i t i a t d amines are extracted from plasma with d i e t h y l ether, separated by TLC and measured i n a s c i n t i l l a t i o n counter. The method i s reported sensitive t o 0.1 mle/L.

7.

Metabolism and Excretion

Dopamine metabolism and T g r e t i o n was studied follawing intravenous administration of C material to six healthy males ( 1 4 ) . Dopamine and its metabolites are excreted primarily in the urine: the rsdioactive dose was quantitatively recovered a f t e r 5 days. Approximately 75% of the dose was converted i n t o dopamine-related lnetabolites. The principal product was 3-methoxy-4-hydroxyphenylacetic acid. Other p r h e n t metabolites were 3-methoxydopamine, 3,4-dihyroxyphenylacetic acid, 3,4-dihydroxyphenylethanol and 3-methoxy-4-hydroxyphenylethanol. The remainbg 25% of the infused dopamine w a s transformed i n t o norepinephrine and a p a r e d i n the urine principally as metabolites of norepinephrine. The conjugates of doparnine arid its metabolites were studied in cultured human skin fibroblastc and rat hepatam c e l l s ( 1 6 ) . These studies along with references c i t e d therein indicate the principal dopamine conjugates a s the 3-0 sulphate, and the 3+glucuronide.

8.

Ackncwledgemnt The manuscript was expertly typed by M s . Martine Bunting.

JAMES E. CARTER E T A L .

212

References

1.

Anon., I n t r o p i n product literature, Awrican Critical C a r e , WGaw Park, IL 60085.

2.

The Merck I d a , 9 t h Edition, 3422, Merck & Co., Inc., F?ahway, N J , 1976.

3.

S. P a l e d , Walter C. Mccrone Associates, Inc., Chicago, I L , 60616.

4.

Personal Comnunication.

W i l l i a m J. McGranahan, Northwestern University, D e p r t m n t of Chemistry, Evanston, I L 60201. Personal

Cammication. 5.

Hoying L. Hung, Northvestem University, Departrent of Chemistry, Evanston, I L 60201. Personal C m i c a t i o n .

6.

Paul W. Erhardt, American Critical C a r e , McGaw Park, IL, 60085. Personal C a m m i c a t i o n .

7.

P. Fabre, French P a t e n t 2332748-y36,

8.

Anon., United States Pharmacopeia, XX Revision, 919,

(1977).

Mack Publishing Co., Easton, PA, 1980. Doty, Anal. Chem., 20, 1166 (1948). --

9.

J.R.

10.

P.A. A s r m s and C.R. (1979).

meed, J. Chromatogr.,

169, 303

11. L.A. Gardella, J.F. Zaroslinski and L.H. Possley, Am. J. Hosp. Phann., 32, 575 (1975). - 12. 13.

L.A. Gardella, H. Kesler, J.E. C a r t e r and J.F. Zaroslinski, Am. - -J. Hosp. Phann., 33, 537 (1976). L.A. Gardella, H. Kesler, A.H. Amann and J.E. 35, 581 (1978).

- -J. Hosp. Pharm., Am.

Carter,

14. M. Goodall and H. Alton, Biochem. Pharmacol., 17, 905 (1968). 15.

G. Koch, U. Johansson and E. Arvidsson, J. C hm. -- l h . C C l h Biochem., 18, 367 !1980).

16.

P.A. Crooks, X.O. Breakefield, C.H. Sulens, C.M. C a s t i g l i o n e and J . K . Caward, Biochem. J., __ 176, - 187 (1978).

ERGONOVINE MALEATE Van D.Rdj

1. Description 1.1 General Classification

2.

3. 4. 5. 6.

I. 8.

1.2 Name, Formula, Molecular Weight 1.3 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectra 2.2 Ultraviolet Spectra 2.3 Fluroescence and Phosphorescence Spectra 2.4 Nuclear Magnetic Resonance Spectra 2.5 Mass Spectrum 2.6 Differential Scanning Calorimetry 2.7 Melting Range 2.8 Crystal Properties 2.9 Solubility 2.10 Optical Rotation 2.11 Circular Dichroism Configuration Synthesis 3.1 Biosynthesis 3.2 Chemical Synthesis Stability Identification Methods of Analysis 6.1 Elemental Analysis 6.2 Direct Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Titrimetric Analysis 6.5 Automated Analysis 6.6 Fluorometric Analysis 6.7 Chromatographic Analysis 6.8 Radiochemical Procedures Metabolism References

Analytical Profiles of Drug Substances Volume 11

273

274 274 274 274 275 275 275 275 279 283 283 283 283 283 289 289 289 289 290 290 293 293 293 293 293 293 295 295 295 296 296 308

Copyright 0 1982 by The American Pharmaceutical Aamiation

ISBN 012-260811-9

VAN D.REIF

274

Description 1.1 General Classification Ergonovine is a naturally occurring alkaloid found in ergot (Claviceps purpurea). It is classed as one of the water-soluble, amine ergot alkaloids, and is an orally-active oxytocic (1,2). The maleate salt exhibits greater stability than the free base and is the usual form in which the alkaloid is utilized (3). 1.

1.2 Name, Formula, Molecular Weight The name used by Chemical Abstracts for ergonovine maleate is [8,&(S)]-9,10-didehydro-N-(2-hydroxy-lmethylethyl)-6-methylergoline-8-carboxam~deI (Z)-2-butenedioate (1:l) salt. The Chemical Abstracts Registry Number is 129-51-1. Ergometrine, ergostetrine, ergobasine, ergotocine, and D-lysergic acid L-2-propanolamide are other names that have been used for this alkaloid (2,4). H I

HC-COOH It

HC-COOH

c 1gH23N302 ' CqH404

Mol. Wt. = 441.48

1.3 Appearance, Color, Odor Ergonovine maleate is a white to greyish white, odorless, crystalline powder.

ERGONOVINE MALEATE

2.

275

Physical Properties 2.1

Infrared Spectra The infrared spectra of ergonovine maleate in potassium bromide and in mineral oil are given in Figures 1 and 2 , respectively. Both were recorded on a PerkinElmer 467 Grating Spectrophotometer. The KBr spectrum is similar to that previously published (5). The KBr procedure is also used for an official identity test (6). Structural assignments (7,8) of some of the significant bands are given in Table I. As reported (71, a carbonyl band for the maleate moiety is not distinguished in the KBr dispersion, and is seen only as a shoulder at 1690 cm-l in the mull. Table I Infrared Assignments for Ergonovine Maleate (0.5% KBr Dispersion) Frequency range (cm-l)

Assignment

3260-3500

N-H, 0-H stretch

1650

amide C=O stretch

1570

carboxylate annion

1055

primary hydroxyl

750,775

indole C-H

2.2 Ultraviolet Spectra The ultraviolet spectrum of ergonovine maleate in ethanol is shown in Figure 3 . An absorptivity o f 20.5 (E=9160) was determined for the maximum at 311 nm in alcohol. A similar spectrum is obtained using water as solvent; the absorptivity at the maximum at 311 nm was found to be 18.7 (E=8260). The absorptivity values agree with those previously published (9,101. 2.3 Fluorescence and Phosphorescence Spectra A fluorescence emission spectrum in ethanol is shown in Figure 4 . Excitation was at 3 2 5 nm. It was obtained on a Perkin-Elmer Model 512 fluorescence spectrophotometer. A similar spectrum was reported by Bowd et al. (10) for ergonovine, and phosphorescence maxima were found at 514,554, and 611 nm at 77OK in ethanol.

WAVELENGTH (Microns) 2.5

3.0

I

I

4OoO

I

3500

4.0

I

m

6.0 1

5.0 I

8.0

10.0

I

I

I

I

I

i

i

2500

2Ooo

1800

1600

1400

1200

loo0

12.0

20.0

16.0

i

800

i

600

cm-4

WAVE NUMBER

Figure 1

- Infrared Spectrum of Ergonovine Maleate, USP Reference Standard, Lot L(KBr Pellet)

30.0 40.0 1.0

i

400

200

WAVELENGM (Microns)

2.5 1

3.0

4.0

5.0

I

1

I

6.0

7.0

I

1

8.0 I

9.0 I

10 ,

12 I

14 1

M

16 18 20 I

I

I

w

V

z U

E h)

21 21

5, z U e I z

c

7

800

600

WAVE NUMBER(Crn-4

Figure 2

-

I n f r a r e d S p e c t r u m o f E r g o n o v i n e Maleate, USP R e f e r e n c e S t a n d a r d , Lot L (Mineral O i l Mull)

400

40 I

VAN D.REIF

278

0.7

0.6

0.5

y

z a

0.4

m ac

Ln 0

$

0.3

0.2

0.1

0

1

1

210

1

1

1

1

Fig. 3. Ultraviolet Spectrum of Ergonovine Maleate (USP Reference Standard, Lot L ) . Solvent-Alcohol.

1

235 260 285 310 335 360 WAVE LENGTH IomJ

I

560

I

500

1

I

440 380 NANOMETERS

1

320

Fig. 4. Fluorescence E m i s s i o n Spectrum of Ergonovine Maleate, 0 . 1 mg/ml (USP Reference Standard, Lot L)

279

ERGONOVINE MALEATE

2.4 Nuclear Magnetic Resonance Spectra The proton NMR spectrum of ergonovine maleate (USP Reference Standard, Lot L ) in deuterated dimethylsulfoxide (d6) with tetramethylsilane as internal standard is shown in Figure 5. The spectrum was obtained with a 100 MHz Varian XL-100 spectrometer (11). Spectral assignments are given in Table 11. Bands corresponding to side chain hydrogens were identified by irradiation of the side-chain methine hydrogen (s=3.9). This resulted in conversion of doublets at 1.12, 3.44, and 8.25 ppm to singlets. The alcohol hydrogen was assigned to the shift at 3.68 ppm because resonance was lost at this shift after D20 exchange. The shift at 10.98 ppm, assigned to the indole-NH, also showed a partial loss of resonance after D20. Similarly, the only resonances above 7.5 ppm for related indoles, 8.5 ppm (CDC13) for lysergic acid dimethylamide and 11.4 ppm (deuteropyridine) for penniclavine, were attributed to the indole-NH (12,13). The 6-NCH3,H8,H9, and aromatic proton assignments are also similar to those reported for penniclavine and lysergic ac id dime thy1amide . Table I1 Proton NMR Spectral Assignments for Ergonovine Maleate Chemical Shift ( S )

Multiplicity

Assignment

1.12

doublet

-CH3 (side chain)

3.10

singlet

6-NCH3

3.44

doublet

C-CH20

3.68

singlet

C-OH

3.9

mu1 t ip let

NCH-C

4.30

mu1 t ip let

c(H-8

6.14

singlet

maleate C-H

6.57

sharp multiplet

H-9

7.6-7.4

mu 1tip le t

H-12,H-13,H-14,H-2

8.25

doublet

8CNH

10.98

singlet

indole-NH

28 1

ERGONOVINE MALEATE

The 13C NMR spectrum of ergonovine maleate (USP Reference Standard, Lot L) in deuterated dimethylsulfoxide is shown in Figure 6. It was recorded on a Varian FT-80-A spectrometer at ambient temperature (11). The spectral assignments are given in Table 111. The chemical shifts are similar to those reported by Bach et al. (14) for ergonovine. Table 111 13C NMR Assignments for Ergonovine Maleate Carbon

E !

-C02H

169.3

-CON

168.1

HC=CH

136.2

c-10

134.6

C-15

131.8

C-16

126.0

c-11

125.7

C-13

123.3

c-9 c-2

121.0

C-14

112.5

c-12

111.6

c-3

106.8

OCH2 c-5

65 .O 61.8

c-7

53.6

HN-CH

47.7

N-CH3

41.7

C-8

40.8

c-4

24.7

-CH3( side chain)

17.7

120.5

ERGONOVINE MALEATE

283

2.5

Mass Spectrum The mass spectrum of ergonovine maleate was obtained with a Kratos DS-50s Data System coupled with a MS-902 double focusing, high resolution mass spectrometer (15). The ionizing electron beam energy was at 70eV and the probe temperature was 250°. A line graph of the mass spectrum is shown in Figure 7 and identification of the pertinent masses are presented in Figure 8. The retroDiels-Alder reaction fragments at m/e 282 and 196 and the fragments at m/e 220-223 were postulated by Bellman (16) for other lysergic acid analogs. The identity of fragments at m/e 167 and 154 have been postulated for other ergot alkaloids (17,181. 2.6 Differential Scanning Calorimetry The DSC thermogram (19) of ergonovine maleate (USP Reference Standard, Lot L) is shown in Figure 9. The thermogram was obtained at a heating rate of loo/ min in a nitrogen atmosphere using a Perkin-Elmer DSC-2. No endotherms or exotherms were observed other than that associated with the decomposition melt. 2.7 Melting Range The melting range must be taken under a protective environment to limit decomposition. The following melting point temperatures have been reported for ergonovine maleate: OC 167 (with decomposition) 180-190 (in paraffin) 187-191 (silicone oil) 186-196 (under nitrogen) 185-190 (sealed capillary)

Reference

4 20 21 21 21

2.8

Crystal Properties The X-ray diffraction pattern of ergonovine maleate (USP Reference Standard, Lot L) as obtained with a Philips diffractometer using C u K d radiation (191, is presented in Figure 10. The calculated "d" spacings are listed in Table IV. 2.9

Solubility The following solubilities have been reported for ergonovine maleate (4,221:

53111SNllNI 3hllVl3tl

m/e -

RELATIVE INTENSITY

ASSIGNMENT

325 310 307 294

100

m+

mt -CH3

0.4

2.2 2 .o

mt-H$ m t -CHflH

282

223

25.5

222

6.8

221

196

[

167

&NH]'

[ & 1'

154

Figure 8

- Mass Spectrum Fragments 285

7.3

6.7

VAN D.REIF

286

t

0 c3

z

w

~

50

75

100

125

150

175

200

OC

F i g . 9. D i f f e r e n t i a l Thermal A n a l y s i s Spectrum of Ergon o v i n e Maleate (USP R e f e r e n c e S t a n d a r d , L o t L)

225

loo

-

90 8070 60-

c

B z

50-

I-

40-

29

Figure 10

- X-ray Diffraction Pattern of Ergonovine Maleate (USP Reference Standard, Lot L)

VAN D.REIF

288

Table I V

X-Ray Powder Diffraction Pattern 2 0

d (Ao)

1/10

39.6

2.28

4

38.7

2.33

4

36.5

2.46

11

34.4

2.61

4

32.8

2.73

4

29.6

3.02

7

28.9

3.09

13

26.4

3.38

26

24.8

3.59

19

23.9

3.72

24

22.9

3.88

20

22.1

4.02

32

21.6

4.11

98

19.9

4.46

16

18.6

4.77

18

17.7

5.01

13

16.2

5.47

64

15.1

5.87

40

14.2

6.24

89

8.8

10.00

100

7.5

12.00

36

ERGONOVINE MALEATE

289

Solvent water alcohol chloroform ether

Solub i 1ity (mg/ml) 28 8 nearly insoluble ( <0.1) nearly insoluble ( < 0.1)

2.10 Optical Rotation The following optical rotations were reported (23) for ergonovine maleate:

[d]i0= +53 (cz1.0 in water)

20 [a1578 = +57 (Cz1.O in water) 20 ["(I546 = +74 (C=l.O in water) 20 [HI436 = +252 (C=l.O in water)

USP material ( 6 ) meets the specification, [O(

Ii5"

= +51 to +56 ( G 0 . 5 in water).

2.11 Circular Dichroism-Configuration A circular dichroism spectrum was reported (24) for ergonovine maleate. Maxima were at approximately 218 and 318 nm, and a minimum was at 248 nm. Other isomers containing the D-lysergic acid moiety gave similar spectra; a compound containing the L-lysergic acid moiety showed a reversed effect. This is consistent with the positive Cotton above 300 nm reported for D-lysergic acid (25). The rotatory dispersive effects are due to the configuration at C-5; the configuration at C-8 has only an additive or subtractive effect. In the case of Dlysergic acid, and therefore, ergonovine, the hydrogen at C-5 is in t h e 4 position (25) and the C-5 center is in the S configuration (26). 3. Synthesis 3.1 Biosynthesis Ergonovine was originally isolated from ergot extracts (2,27-29). It has since been biosynthesized in cultures of Claviceps species (30-351, and has been isolated from seeds of I omoea violacea (36,371 and Argyreia nervosa (38 The following pathway has been constructed (39-44) for biosynthesis of the ergoline skeleton by Claviceps species:

+

VAN D.REIF

290

mevalonic acid + tryptophan

elymoclavine

5.

+

) -

agroclavine

,<-

6-methyl-Ll 8,9-ergoline-8-carboxylic

4-dimethylallotryptophan chanoclavine-I acid

\1

lysergic acid The final step to produce ergonovine was shown to involve incorporation of alanine with lysergic acid (44). Chemical Synthes.is Various semi-synthetic procedures for ergonovine maleate involve addition o f the alkyl side chain to lysergic acid. The D-lysergic acid starting material is obtained by alkaline hydrolysis of an ergot alkaloid fraction of biosynthetic origin (45). A relatively high-yield sequence is shown in Figure 11. Lithium lysergate is reacted with a sulfur trioxide-dimethylformamide complex at O°C. L-2-amino-1-propanol is added, and the product is isolated after addition of water and treatment with maleic acid. Procedures for formation of the amide bond using lysergic acid chloride ( 4 6 ) or phosgene (47) have been patented. A complete fiftegn-step synthesis o f the D-lysergic acid starting material from 3b-carboxyethylindole has also been reported (48). 3.2

4.

Stability

The maleate salt shows improved stability over ergonovine base, but the salt also oxidizes and darkens in the presence of oxygen (3). The stability of ergonovine s o l u tions has been improved by the use of antioxidants such as ascorbic acid (491, methionine, and d-mercaptopropionylglycine (50). The known degradation products are shown in Figure 12. Heat treatment of ergonovine (a dextrorotatory isomer) under acid or alkaline conditions causes a reversible isomerization at the 8-position to form the dextrorotatory ergonovinine, also known as isoergonovine. More stringent acid or base treatment eventually results in hydrolysis to lysergic acid, isolysergic acid, and 2-amino1-propanol (51,521. When ergonovine is refluxed in the

LiOH-HZO

D-LYSERGIC ACID

CH30H

CO NH

-c

w

-CH3

DMF CH3CHNH2 I

CH20H

I

MALEIC A C I D

ERGONOV I NE MALEATE Figure 11

-

Synthesis of Ergonovine Maleate from Lysergic Acid

VAN D.REIF

292

y CONH-C-CH~ A,

H+

or OH^

1

OH{

ERGONOVINE

d3 & &

ERGONOVlNlNE (Iso-ergonovine)

OH * ._

HN

LYSERGIC ACID

y

y

CONH-C-CH3

H;HzoH

ISO-LY SERG IC ACID

CONH-C-CH3

+

HN

H;HpH

HN LUMIERGONOVINE II

LUMIERGONOVINE I

Figure 12

- Degradation of E r g o n o v i n e

ERGONOVINE MALEATE

293

presence of amines (53), the centers at C-8 and C-5 are both racemized to give mixtures of ergonovine, ergonovinine, L-lysergic acid L-2-propanolamide and L-isolysergic acid L-2-propanolamide. When acidic solutions of ergonovine are irradiated, the 9,lO double bond is hydrated to form lumiergonovine I and small quantities of lumiergonovine I1 (54-56). Degradation of the ergonovine indole ring has been indicated by reports (57) of unidentified fluorescent degradation products which do not react with p-dimethylaminobenzaldehyde. 5.

Identification

Alternately known as Van Urk's (58) or Erhlich's (59) reagent, 1-dimethylaminobenzaldehyde has had widespread use in ergonovine identity tests (59-61) and chromatographic detection sprays (6,60,62). Indoles give a blue color with this reagent (61); phenols and amines may also react, usually to give other colors (58,591. Color reactions may also be produced with aldehydes such as vanillin, piperonal, and paraldehyde (63). Iodinated potassium iodide (brown flocculent) and ferric chloride in phosphoric acid at 80° (blue-violet color) are two additional identity test reagents (60). 6. Methods of Analysis 6.1 Elemental Analysis The elemental analysis of ergonovine maleate (USP Reference Standard, Lot L) is presented below. Element

% Calculated

62.57 6.17 9.52

% Reported (64)

62.55 H 6.03 N 9.38 6.2 Direct Spectrophotometric Analysis Bayer (65) described an ultraviolet spectrophotometric method for ergonovine maleate tablets. The tablets are extracted with 1% tartaric acid and the absorbance is measured at 317 nm. C

VAN D.REIF

294

Colorimetric Analysis Official assays for ergonovine maleate drug substance, tablets, and injection (6,601 employ the p-dimethylaminobenzaldehyde reagent. Drug substance or injection samples are dissolved in water; tablet samples are dissolved in aqueous tartaric ac id-benzalkonium chloride. The reagent is dissolved in aqueous sulfuric acidferric chloride. After color development, absorbances are read at 555 nm. Light or hydrogen peroxide (63,66,67) have also been used in place of ferric chloride to catalyze color development for quantitation. A similar quantitative procedure using sodium nitrite t o catalyze color formation was shown to have the advantages of speed, sensitivity, and color stability (62). 6.3

The specificity of the reaction for indoles under several conditions was studied by Kupfer (61). The specificity was very dependent on the particular conditions employed and the development times. In general, for indoles to be reactive, an unsubstituted 2- or 3- position was required. The mechanism proposed by Pohm (68) involved attachment of one aldehyde molecule at the 2-positions of two indole molecules with release o f water. An assay for ergonovine maleate tablets using metaldehyde to produce a color at 550 nm has been published (69). A colorimetric assay for tablets using ion-pairing with bromocresol purple was reported (70). 6.4 Titrimetric Analysis Ergonovine maleate can be determined titrimetrically in glacial acetic acid with a 0 . 0 5 N perchloric acid titrant and crystal violet indicator (71,721. The NF XIV drug substance assay (73) is similar, except that acetic anhydride is added to the system. Ergonovine was determined in mixtures after thin-layer chromatography with chloroform-methanol (97:3) on alumina. Spots were eluted with chloroform, acetone was added, and the mixture was titrated with 0.005 N perchloric acid in methanol (74). The maleate moiety has been determined with 0.01 potassium methoxide titrant, thymol blue indicator, and pyridine as solvent (71).

ERGONOVINE MALEATE

295

6.5 Automated Analysis Kirchhoefer and Wells (75) developed automated methods for ergonovine maleate tablets and injections. Samples were dissolved in pH 6.0 tartrate buffer. For colorimetric quantitation, the sample stream was made basic and extracted with n-butanol. The extract was mixed with p-dimethylaminobenzaldehyde reagent, and after reaction, the stream was mixed with sodium nitrite for determination at 550 nm. For fluorometric detection the sample stream was diluted with tartrate buffer. Excitation was at 325 nm and the emission was read at 432 nm. The methods were tested for interference from common tablet and injection excipients. Robertson et al. (76) reported a general procedure for basic drugs in which ergonovine was included. Bromthymol blue solution was mixed with an aqueous buffered sample stream. The ergonovine-dye complex was extracted into chloroform and determined at 410 nm. 6.6

Fluorometric Analysis Fluorometry has been used to determine ergonovine maleate in tablets and injections in an automated procedure (75) (see section 6.51, and after column chromatography (77) (see section 6.73). Samples were read in 0.1 tartartic acid buffer with excitation and emission at 325 and 432 nm, respectively. 6.7 Chromatographic Analysis 6.71 Thin-Layer Chromatography The thin-layer chromatographic systems used to analyze ergonovine and ergonovine maleate are listed in Table V. The applications, means of quantitation or detection,and procedure specificity are listed when this information was supplied in the literature sources. The last five systems employ ion-exchange layers. 6.72 Paper Chromatography Paper chromatographic systems for ergonovine and ergonovine maleate are given in Table VI. Applications and detection agents are also listed. Sprince (100) described a variation of the Ehrlich reagent which gave rapid, long-lasting color development for paper chromatography. A p-dimethylaminobenzaldehyde in HCl spray was followed by a 1% sodium nitrite spray.

296

VAN D.REIF

6.73 Column Chromatography The column chromatographic methods that have . been used to analyze ergonovine maleate or ergonovine base are described in Table VII. 6.74 Gas Chromatography Sondack (103) analyzed ergonovine maleate in tablets and injectables after derivatization with N-trimethylsilyldiethylamine and N-trimethylsilylimidazole. A 1% OV-1 on Gas Chrom Q column at 260° was used. The separation of ergonovinine and lumiergonovine I from ergonovine was demonstrated. 6.75 High-Performance Liquid Chromatography High-Performance Liquid Chromatographic systems for ergonovine and ergonovine maleate are listed in Table VIII, along with applications and specificity informat ion. 6.76 Electrophoresis The electrophoresis of ergonovine on cellulose thin layers at 500V and 3000V was reported (112). The following electrolytes were found to be the most suitable: formic acid-acetic acid-water (26:120:1000), ammonia-water (2:98), and ammonia-water (1:9). A high voltage paper electrophoretic separation of ergonovine from its diasteromer, D-lysergic acid D-propanolamide, was reported (113). 6.8

Radiochemical Procedures A radioimmuno assay for ergonovine and other

ergot alkaloids in plasma was reported (114). Detection was at the picomole level from 4 ml of plasma. The antisera, produced by a conjugate of lysergic acid with human serum albumin, reacted with lysergic acid derivatives, but not with tryptophan. An antibody produced by a mixed anhydride reaction of lysergic acid with bovine serum albumin reacted with simple lysergic acid derivatives and some clavines (115). A similar coupling using a Mannich reaction yielded an antibody specific for lysergic acid. 7. Metabolism Two metabolites of ergonovine, identified as the 6-glucuronides of 12-hydroxyergonovine and 12-hydroxyergonovinine, were excreted in the bile of rats (99). Two other metabolites were tentatively identified as glucuronides of ergonovine and ergonovinine. An earlier report (116) demonstrated the formation of two unidentified polar metabolites of ergonovine in rats.

Table V Thin-Layer Chromatography Systems Appl icat ion (Quantita t ion/De tec tion

Separation of Ergonovine from: Ergonovinine , ergot alkaloids

Ch 1oro form-Methano1 (97:3)/alumina

Quantitation of ergonovine in ergot mixtures (colorimetric) Quantitation of ergonovine in tablets,injections (in situ fluorometric) -Quantitation of ergot mixtures (titrimetric)

Ethyl Acetate-n-HeptaneDiethylamine (7:6:O .0005 ) / Cellulose-formamide Chloroform-Ethano1Ace tone (6:4:4)/silica gel G

Toluene-Butanol (4N HC1 saturated) (6:4)/silica gel

78

-

79

Ergotamine, ergocryptine, ergocrystine

74

Quantitation of ergot mixture s (in situ fluorometric) --

8 Ergot alkaloids

80

23

Ergot alkaloid quantitation (colorimetric)

Ergonovinine , 10 ergot alkaloids

81

5

Ergot alkaloid quantitation (colorimetric)

Ergonovinine, 10 ergot alkaloids

81

Chloroform-Methanol (3:l)/silica gel

Chloroform-E thanol (9:l)/silica gel G

Ref. -

Table V (continued) Solvent/Plate

Rf x 100

Application (Ouantitation/Detection)

Separation of Ergonovine from:

Ref. -

Ethyl Acetate-DMF17 Ethanol (13:1.9:0.l)/silica gel

Ergot alkaloid quantitation (colorimetric)

11 Ergot alkaloids

82

Benzene-DMF (13:2)/ silica gel

12

Ergot alkaloid quantitation (colorimetric)

11 Ergot alkaloids

82

Methanol-Acetic AcidWater (2:1:2)/silica ge 1 Methanol-Water-Tartaric Acid (40:60:l)/silica ge 1 Benzene-Ch lor0formEthanol (2:4:1)/ silica gel Chloroform-MethanolWater (75:25:3)/ silica gel

30

Ergot alkaloid quantitation (colorimetric)

Ergotamine, agroclavine, chanoclavin

83

41

Ergot alkaloid quantitation (colorimetric)

Ergotamine, agroclavine, chanoclavin

83

11

Ergot extract quantitation (colorimetric)

8 Ergot alkaloids,

84

-

Chloroform-Acetone45 Methanol-Triethylamine 15 :10:5 : 1 / s i 1ica ge 1

ergonovinine

Purity determination and Identity for Ergonovine maleate (visual after p-dimethylaminobenzaldehyde) Identification of Impurities in Ergonovine maleate (UV)

Ergonovinine,related a 1kalo id s

24

Table V (continued) Application Separation of (Quantitationstection) Ergonovine from:

6 Systems/silica gel, alumina, cellulose

-

~

Ref.

Ergot Alkaloid Identifi- Ergonovinine, cation ( 10% CuSO4-2% 5 ergot alkaloids ammonium hydroxide, 5:l)

85

34 Systems /silica 0 to 80 gel, alumina

Alkaloid Identification in I. violacea extracts (UV,p-dimethylaminobenzaldehyde + sodium nitrite

Ergonovinine, LSD, iso-LSD

86

0 to 66 17 Systems/silica gel, alumina, cellulose

Ergot Alkaloid IdentifiEhrlich's cation ( W , Reagent)

14 ergot alkaloids

87

LSD Identification

Ergonovinine, 20 ergot alkaloids

88

Ergonovinine, 20 ergot alkaloids

88

(Ehrlich's Reagent) LSD Identification

9 alkaloids

89

15 ergot alkaloids

90

Chloroform-Methanol (9:l)/silica gel GF

10

Chloroform (NH3 saturatedl-Methanol (18:l)/silica gel GF

13

TrichloroethaneMethanol (9:1)/ silica gel

21

Diisopropyl EtherTHF-Toluene-Diethylamine ( 7 0 : 15 :15:O. 1 ) / si1ica ge1- formamide

(Ehrlich's Reagent) LSD Identification

(Ehrlich's Reagent)

0

Ergot alkaloid separation (w)

Table V (continued) Solvent/Plate

Rf x 100

Acetone/O.lM 78 Ammonium Carbonate/ Ethanol (32.5: 67.5:l)/silica gel G 11 Systems/silica gel, alumina

0-83

Application

Ref. -

(Quantitation/Detection)

Ergonovine from:

Determination of Ergotoxine Alkaloids (vani11in/H2SOq )

Ergonovinine, 13 ergot a1kaloids

91

methylsergide, methergine detection

methylsergide, methergine

92

Ergot alkaloid identification (0.5% chlorimino-2,6-

4 Ergot alkaloids

93

Chloroform-BenzeneMethanol (formamide saturated,50 :4: lO)/silica gel G

-

Acetic Acid-HC1 (pH 0.6-2.35)/ alginic acid-cellulose, Rexyn 102cellulose

36-4

Alkaloid identificat ion (6M acetic acid + UV, Dragendor ff ' s )

30 alkaloids

94

15

Alkaloid identification (6M acetic acid + UV, Dragendorf f ' s )

30 alkaloids

94

1M HC1 in Water21 EFhanol ( 1 : 4 ) / Dowex 50-X4-cellu lose

Alkaloid identification (6M acetic acid + UV, Dragendor ff ' s )

30 alkaloids

94

1M Acetic Acid in Water-E thanol (7:3)/Rexyn 102cellulose

dichloroquinone/methanol;

0.3% Congo Red, pH 7; 0.5% quinone/aq. HC1)

Table V (concluded) Solvent/Plate

Rf x 100

Application -(Quantitation/Detection)

Separation of Ergonovine from:

Ref. -

0.5M Acetate/AG 1~4 Tacetatelcellulose

21

Alkaloid identification (6E acetic acid + UV, Dragendor f f ' s

30 alkaloids

95

0.5M Ammonium Aceta te/DEAE ce 1 lu 1o se

49

Alkaloid identification (6M acetic acid + UV, Dragendor f f ' s

30 alkaloids

95

Table VI Paper Chromatography Systems for Ergonovine Stationag Phase

Mobile -

Application

Separations

Detection

Reference

Water-insoluble UV light from water-solble ergot alkaloids, ergometrine from ergometrinine

n-Butanol-Acetic Whatman No. 1 (descending) Acid-Water (4:1:5)

Estimation of ergonovine maleate in ergot extracts and injections

Whatman No. 3 5% octanol (ascending)

Ace tone-5% Aqueous Ammonia (2:3)

determination of ergonovine in ergot extracts

A.H. Thomas NO. 3677pH 6 sodium hydroxidepot assium pho spha te

n-ButylacetateNitromethaneEthyleneglycolmonoethylether Ace tate-Pyr id ine Water (100 :50: 5 0 : 8:lO)

determination of ergonovine in water-solble ergot extracts

Paper loaded with 10% d imethy 1 phthalate in chloroform

Formamide-O.1N Pota s s ium Hydroxide (1:4)

limit test for impurities in ergonovine maleate

-

ergonovine from ergonovinine

-

p-d imethylaminobenzaldehyde

uv

p-d imethylaminobenzaldehyde

96

97

98

60

Table V I (continued) Stationary Phase

W

E:

Mobile

-Application

Separations

Detection ~-

uv

Whatman No. 1 or No. 3

Butanol (water saturated)

metabolite ident i f ication

Ergonovine from 12-hydroxyergonovine, metabolites

Ehrlich ' s Reagent

Whatman No. 1 or No. 3

Butanol metabolite iden(ammonia, tification water saturated)

Ergonovine from 12-hydroxyergonovine, metabolites

Ehrl ich ' s Reagent

9

uv ,

Reference

99

99

Table VII Column Chromatography for Ergonovine Packing

Eluent

App 1ication

Separates Ergonovine from -

Detec t ion

Ref.

TLC

24

Silica gel

ChloroformAcetone-Methsno 1-Tr iethylamine ( 15 : 10: 5:l)

Impurity Isolation

Ergometrinine, D-LSAD-2-propranolamide male ate

Sephadex LH-20

96% Ethanol or DMF or Ace tone

Plant Extracts

5 Ergot Alkaloids

Van Urk Reagent

101

Sephadex G 25

Water

Plant Extracts

Lysergic Acid

Van Urk Re agent

101

Ether Ce 1ite I Sodium Bicarbonate

Assay Tablets Injections

Celitel Water

Degraded Extracts

Powdered Ergot Extracts

Butanol-Benzene-20% Acetic Acid (1:1:2)

Cel itel (1)Chloroform Citric Acid (2)Sodium Bicarbonate ( 3 )Chloroform

Fluorescence

77

Lumiergonov ine I and I1

Van Urk Reagent

56

Water-Insoluble Ergot Alkaloids

p-Dimethylamino benzaldehyde

98

Table VII ( c o n t i n u e d ) -Packing

Cellulose/ pH 3 . 0 CitratePhosphate

W

E:

Eluent

(110.1% p y r i -

-Application

Ergot Alkaloid dinelether Mixtures (2110% d i e t h y l amine / e t h e r (3)ether

parates -S e_

Ergonovine

~Detection

Re€. -

from -

Water I n s o l u b l e E r g o t A l k a l o i d s , Ergonovinine

p-Dimethylamino b e n z a l dehyde

102

Table VIII High-Performance Liquid Chromatography Systems for Ergonovine Co 1umn

Mobile

Application

Ergonovine Separates from -

Detection

Ref.

Ergonovinine,Lysergic Acid

312 nm

104

AcetonitrileIdentification 0.01M Ammonium of Components of Carbonate (2:3) Plant Extracts, Fermentation Mixtures

Ergonovinine, 11 Ergot Alkaloids

320 nm

105

LiChrosorb

n-Hexane-

Ergonovinine, 11 Ergot A1 kaloid s

105

ChloroformEthanol(4:4:1)

Identification of Components of Plant Extracts, Fermentat ion Mixtures

320 nm

SI-60

Corasil C18

Methanol-0.1% Ammonium Carbonate (3:2)

Assay of Illicit LSD Preparations

Ergot Alkaloids

Corasil 11

AcetonitrileIsopropyl Ether (1:3)

Assay of Illicit LSD Preparations

Ergot Alkaloids

254 nm

108

microBondapak C18

Methanol-AceForensic Mixture tic Acid-0.005 Identity M Heptane Sulfonic Acid (40:1:59)

Ergot Alkaloids

254 nm

109

microBondapak C18

Acetonitrile1% Acetic Acid (1 :4)

LiChrosorb RP-2, Rp-8 and RP-18

Injection, Tablet Assay

Fluorometric 107

Table VIII (continued) High-Performance Liquid Chromatography Systems for Ergonovine App 1ic at ion -

Ergonovine Separates Detection

Ref.

Separation o f Ergot Mixtures

Ergonovinine, 15 Ergot Alkaloids

310 nm

110

Separation of Ergot Mixtures

Ergonovinine, 15 Ergot A1 ka loid s

310 nm

110

Chloroform-Iso- Separation of Ergot Mixtures propano1 (9:l)

Ergonovinine, 15 Ergot Alkaloids

310 nm

110

LiChrosorb NH2

Diethyl EtherEthano 1 (93:7)

Separation of Ergot Mixtures

Ergonovinine, 15 Ergot Alkaloids

310 nm

110

LiChrosorb

Hexane-ChloroSeparation o f f o rm-Ac e toErgot Mixtures ni tr ile-Methanol (55 :20:25 :3)

Ergonovinine, 10 Ergot Alkaloids

320 nm

111

Co 1umn

microPak NH2

Mobile Diethyl EtherE thano1 (84:12)

Diethyl EtherIsopropanol (3:2)

SI-60

308

VAN D. REIF

8. References 1. P. Brazeau in "The Pharmacological Basis of Therapeutics,"3rd ed., L.S. Goodman and A. Gilman, Eds., The MacMillan Co., New York (1965) p. 880-882. 2. A. Stoll and A. Hofmann in "The Alkaloids," vol. VIII, R.H.F. Manske, Ed., Academic Press, Inc., New York (1965) p. 729,748. 3. G.E. Foster and G.A. Steward, Quart. J. Pharm. Pharmacol., 21, 211 (1948). 4. "The Merck Iaex", 9th ed., Merck and Co., Inc., Rahway, N.J. (1976) p. 3575. 5. A.L. Hayden, O.R. Samuel, G.B. Selzer, and J.Caro1, J . Assoc. Off. -Anal. Chem., 45, 822 (1962). 6. "The United States Pharmacopza," 20th rev., Mack Publishing Co., Easton, PA (1980) p. 284. 7. R.J.Mesley and W.H. Evans, J. Pharm. Pharmacol., 21, 713 (1969). 8. R.M. Silverstein and G.C. Bassler, "Spectrometric Identification of Organic Compounds," 2nd ed., John Wiley & Sons, Inc., New York (1968 ) p.91,94. 9. M.F. Sharkey, C.N. Andres, S.W. Snow, A. Major,Jr., T. Kram, V. Warner, and T.G. Alexander, J. Assoc. Off. Anal. Chem., 51, 1124 (1967). --J. Chem. 10. A. Bowd, J.B. Hudson, and J.H. Turnbull, -S O ~ . ,Perkin 11, 1312 (1973). 11. Hoffman, B., Wyeth Laboratories, Inc., personal communication. 12. K. Bailey and A.A. Grey, &. J. Chem., 50, 3876 (1972). 13. R.G. Mrtek, H.L. Czespi, G . Norman, M.I. Blake, and J.J. Katz, Phytochemistry, 7, 1535 (1968). 14. N.J. Bach, H.E. Boaz, E.C. Kornfeld, C. Chang, H.G. Floss, E.W. Hagaman, and E. Wenkert, 2. Org. Chem., 39 1272 (1974). 15. m s t e d t , J., Wyeth Laboratories, Inc. , personal communic a t ion. 16. S.W. Bellman, J.-Assoc. 51, 164 - - -Off.Ana1. Chem., (1968). 17. M. Barber, J.A. Weisbach, B. Douglas, and G.O. Dudek, Chem.Ind., 1072 (1965). 18. D. Voigt, S. Johne, and D. Groger, Pharmazie, 2, 10 (1974). 19. Sivieri, L., Wyeth Laboratories, Inc., personal communication.

ERGONOVINE MALEATE

309

20. M. Kuhnert-Brandstatter, A. Kofler, R. Hoffmann, and H.C. Rhi, -Sci. Pharm.,33, - 205 (1965). J., 21. M. Kuhnert-Brandstatter and L. Muller, Microchem. 13, 20 (1968). 22 "The United States Pharmacopeia," 20th rev., Mack Publishing Co., Easton, PA (1980) p. 1133. 23. J. Sagel, Pharm. Weekbl., 107, 119 (1972). 63, 1141 (1974). 24. D.L.Sondack,J. Pharm. Sci., 25. A. Stoll and A. Hofmann, in "The Alkaloids," vol.VII1, R.H.F. Manske, Ed., Academic Press, New York (1965) p . 739-740. 2 6 . C. Chothia and P. Pauling, Proc. Natl. Acad. 3, 1063 (1969). 27. M.S. Kharasch and R.R. Legault, J. Am. Chem. 57, - - SOC., 1140 (1935). 28. H.W. Dudley, Proc. R. SOC. (London), 478 (1935). 29. E.C. Kleidere-. k . C h e m . SOC., 57,2007 (1935). 30. M. Abe, T. Yamano, S. Yamatodani, YTKozu, M.Kusumoto, H. Komatsu, and S. Yamada, Nippon Nogei Kagaku Kaishi, 34, 580 (1960); through Chem. Abstr. 5933098f (1963). 31. Neth. Patent Appl. 6,409,764 (1965); through Chem. Abstr. 63:155096b(1965). 32. V.E. Tyler, Jr., U.S. Patent 3,224,945 (1965); through -Chem. Abstr. 6537330f (1966). 33. E. Borowski, K. Braun, K. Breuel, C. Dauth, and D. Enge, Ger. (East) Patent 130,421 (1978); through Chem. Abstr. 91318360f (1979). -34. Y. Yang, D. Yueh, S. Lu, 2. Huo,Q. Sun, X. Lin,Yao Hseuh Hseuh Pao, 14,316 (1979); through -Chem. A b s E --92:20497v (1980). 35. M.A. Sarkisova, A . Shalagina, and A. Bankovskaya, Mikol. Fitopatol., 2,479 (1979); through Chem. Abstr. 92:126896h (1980). 36. K. Genest.' -J. Pharm. Sci.. 55. 1284 (1966). --,-, 37. A. DerMarderosian and H.W. Youngken, Jr., Lloydia, 29, 35 (1966). 38. J. Chao and A.H. DerMarderosian, J. Pharm. Sci.,62, 588 (1973). 501 39. S . Agurell and E. Ramstad, Tetrahedron (1961). 40. R.M. Baxter, S . I . Kandel, and A. Okany, -Chem. Ind., 1453 (1961). 41. H.G. Floss, U. Hornemann, and N. Schilling, Chem. Comm., 105 (1967). I

s.,

=,

m.,

VAN D.REIF

310

42. J.E. Robbers and H.G. Floss, Arch. Biochem. Biophys., 126, 967 (1968). 43. S . Agurell and J. Lindgren, Tetrahedron Lett., 5127 (1968). Acta. Chem. Scand., 23, 3393 44 U. Nelson and S . Agurell, --(1969). Chem., 24, 368 (1959). 45. W.L. Garbrecht, J. Org. 46. I. Sas, L. Rosca, H. Gavrilescu, G. Tudor, I. Velea, E. Nichiforescu, Ger. Patent 2,344,608 (1975); through Chem. Abstr. 83:28436j (1975). -~ 47. L. Bernardi and B. Patelli, Fr. Patent 1,338,023 (1963); through -Chem. Abstr. 60:3026f (1964). 48. E.C. Kornfeld, E.J. Fornefeld, G . B. Kline, M.J. Mann, J. Am. D.E. Morrison, R.G. Jones, and R.B. Woodward, Chem. S O ~ . , 3087 (1956). -75, 798 49. A. Salomon and R.W. Spanhoff, Pharm. Weekblad, (1938); through Chem. Abstr. 32:93979 (1938). ___ 50. E. Noda, S . Izuhara, Y. Katayama, T. Umehara, Japan. Chem. Abstr. 75:67497j(1971). Patent 71 18,148; through -~ 51. S. Smith. and G.M. Timmis, J. Chem. SOC., 1440 (1936). 52. Ibid., 1166 (1936). 53. M. Semonsky and A. Cerny, Chem. Listy,51, - 592 (1957); through -~ Chem. Abstr. 51:11366a (1957). 54. A. Stoll and W. Schlientz, Helv. Chim.Acta, 38, 585 - ~ (19551. 55. H. Hellberg, Acta. Chem. Scand., g , 2 1 9 (1957). Ibid., 56. -13, 1106959). 57. W.E. Moore, Drug Stand., 27,187 (1959). 66,101 (1929); through 58. H.W. VanUrk, Pharm. WeekbGd., _Chem. Abstr. 23:1717 (1929). -~ 59. P. Cooper, -Pharm. J., 495 (1956). 60. "European Pharmacopoeia," vol.1, Maisonneuve S.A., Sainte-Ruffine, France (1969) p. 289-291. 61. D. Kupfer, Anal. Biochem., 8, 75 (1964). 62. L.E. Michelon and W.J. Kelleher, Lloydia, 26, 192 (1963). 63 N.L. Allport and T.T. Cocking, Quart. -J. Pharm. Pharmacol., >,341(1932). 64. Sellstedt, J . , Wyeth Laboratories, Inc., personal communication. 35 (1958); through 65 J. Bayer, Acta Pharm. Hung., 8, Chem. Abstr. 53:2541 (1959). ___ 66. G.E. Foster, -J. Pharm. Pharmcol., 7, 1 (1955). __ __ I

ERGONOVINE MALEATE

31 1

Public Health 67. M.I. Smith, _ ____ _ _ Rep., 45, 1466-1481 (1930). 68. M. Pohm, Arch. Pharm., 286, 509 7i953); Chem. Abstr. (9 5 5 .) 49:8246c 1 69. H.J. VanDerPol, Pharm. Weekblad., 106, 515 (1971). 70. S. El-Masry, Manuf. Chem. & A e r o s o l e w s , 53 (1978). 71. I. Gyenes and K. Szasz, Magyar Kim. Folyoivat, 61, 356 (1955); through -Chem. Abstr. 52:8457g ( 1 9 5 8 r 72. L. Safarik and V. Bumba, -Pharm. Zentralhalle, 96, 3 (1957); through -Chem. Abstr. 52:14085c (1958). 73. "National Formulary," 14th rev., Mack Publishing Co., Easton, PA (1974) p. 254. 74. V.P.Georgievskii, --Probl. Anal. Khim., 1, 94 (1970); through -~ Chem. Abstr. 74:146428v (1971): 75. R.D.Kirchhoefer and C.E. Wells, -J. Assoc. -O f f . Anal. Chem. .58, 879 (1975). Can. J.Pharm. 76. D.L. Robertson, F. Matsui, W.N. French, . Sci., 7, 47 (1972). 77. K. Kirchhoefer, J. Assoc. O f f . Anal. Chem., 9, 1433 (1978). 78. P. Horak Cesk. Farm., 17, 89 (1968); through Chem. Abstr. 6 8 m 5 6 a 1 9 6 g . 79. J.M.G.J. Frijns, Pharm. Weekbl., 106, 865 (1971). 80. M. Prosek, E. Kucan, M. Katic, an=. Bano, Chromatographia, 9 , 273 (1976). 81. K. Roder ,-E. Mutschler, and H. Rochelmeyer, Pharm. Acta Helv., 42, 407 (1967). -82. J.L. McLaughlin, J.E. Goyan, and A.G. Paul, J._Pharm. __ Sci.,53, 306 (1964). 83. S . Keipert and R. Voigt, J. Chromatogr., 64, 327 (1972). J.Pharm. 84. G.E. Ferraro, S.L. Debenedetti, J.D. Coussio, -Pharmac., 28, 729 (1976). 85. K.C. Guvenand L. Eroglu, Eczacilik. Bul., 10, 53 (1968); through -Chem. Abstr. 69:46680=1968). 86. J.M. Weber and T.S. Ma, --Mikrochim.Acta, I, 217 (1976). 87. R. Fowler, P.J. G O ~ and , D.A. Patterson,J.Chromatogr., 72, 351 (1972). 88. A.R. Sperling, J. Chromatogr. Sci., 266 (1974). 89. L .A. DalCort ivo7J.R. Broich , A. Dihrberg , B . E .Newman, Anal. _ _ Chem., 38,1959 (1966). 90. J. Reichett and S . Kudrnac, J. Chromatogr., 87, 433 (1973). 91. G. Szepesi, J. Molnar, Sz. Nyiredy, Fresenius Z. Anal. Chem., 294, 47 (1979). ~~

VAN D.REIF

312

92. J.R. Bianchine, A. Niec, and P.V.J. Macaraeg, Jr., J. Chromatogr., 31, 255 (1967). 93. K.C. Guven.and TFAttinkurt, Eczacilik. Bul. , 20, 46 (1978); through -~ Chem. Abstr. 89:186116h (1978): 94 L. Lepri, P.G. Desideri, and M. Lepori, J. Chromatogr., 123, 175 (1976). 95. Ibid., 116, 131 (1976). 96. J.Pharm. Foster, J. MacDonald, and T.S. G . Jones, Pharmacol. .~ 1 .SO2 (1949). 97. P . Horak and-S. Kudrnac, Cesk. Farm. , 15,483 (1966); Chem. Abstr. 66379622~(1967). -98. T.G. Alexander and D . Banes, J. Pharm. Sci. , 5 0 , 201 (1961). J. Med. Pharm. Chem., 99. M.B. Slaytor and S.E. Wright, ---5 , 4 8 3 (1962). 100. H. Sprince, J. Chromatogr., 3 , 97 (1960). 101. A. Nikolin and B. Nikolin, Phytochem., 11, 1479 (1972). 102. J .E. Carless, _J . Pharm. Pharmacol. , 5,8% (1953). Sci., 63, 584 (1974). 103. D.L. Sondack, -J. Pharm. 104. D.L. Sondack, -J . C h r o m a t S . , E , 615 (1978). 105. L. Szepesy, I. Feher, G . Szepesi, and M. Gazdag, J. Chrom. , 149, 271 (1978). 106. J. Dolinar, Chromatographia, 10, 364 (1977). 107. I. Jane and B.B. Wheals, J- Chromatogr., 84, 181 (1973). J. Chromatogr. 108. J.D. Wittwer, Jr., and J.H. Kluckhohn, Sci., 11, 1.(1973). 109. I. Lurie, J . Assoc. Off. Anal. Chem., 60, 1035 (1977). 110. M. Wurst, M. Flieger, and Z. Rehacek, - Chromatogr., 174, 401 (1979). J. Chromatogr., 111. G. Szepesi, M. Gazdag, and L. Terdy, 191, 101 (1980). 112 A.S.C. Wan, J. Chromatogr., 60, 371 (1971). 113 I.M. Jakovljzic , R.W. Soutec R.H. Bishara, Chromatographia, 11,23 (1978). 114. T . T . Kleimola. Br. J. Clin. Pharmac.. 6. 255 (1978). Planta Med. , 39, 336 (1980); 115. H . Arens and M.H. Zenk, ~through -Chem. Abstr. 933173798q (1980). 116. M. Slaytor, J.N. Pennefather, and S.E. Wright, Experientia, 15, 111 (1959).

.

I - _ -

,

I

FLUFENAMIC ACID Enrico Abignente and Paolo de Caprarh

1. Description 1.1 Name I .2 Formula, Molecular Weight 1.3 Elemental Composition 1.4 Appearance, Color, Odor, Taste 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Fluorescence Spectrum 2.6 Melting Range 2.7 Solubility, Partition Coefficient 2.8 Crystal Properties, Polymorphism 2.9 Dissociation Constant 3. Synthesis 4. Drug Metabolism and Pharmacokinetics 4.1 Metabolism 4.2 Absorption and Excretion 4.3 Protein Binding 5. Methods of Analysis 5.1 Identification Tests 5.2 Titrimetric Analysis 5.3 Colorimetric Analysis 5.4 Spectrophotometric Analysis 5.5 Fluorometric Analysis 5.6 Indirect Atomic Absorption Analysis 5.7 Chromatographic Analysis 6. Determination in Body Fluids and Tissues 7. Acknowledgement 8. References

Analytical Protilcs of Drug Subsurncea Volumc I I

313

314 314 314 314 314 314 314 314 316 320 320 322 323 325 326 327 328 328 328 33 1 332 332 333 333 333 334 335 336 342 343 343

Copyright 0 1982 by The American

PharmaceuticalAssociation ISBN 0-12-260811-9

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

314

1. D E S C R I P T I O N 1. 1 . N a m e F l u f e n a m i c a c i d is d e s i g n a t e d b y C h e m i c a l A b stracts s i n c e 1972 a s 2 - [ [ 3-(trifluoromethyl)phenyl]amin o l b e n z o i c a c i d , w h e r e a s b e f o r e 19 7 2 i t w a s n a m e d N(a,a , a-trifluoro-m-toly1)anthranilic a c i d . O t h e r n a m e s a r e : N - ( 3-trifluoromethylphenyl)anthranilic a c i d , a n d 3'-trifluoromethyldiphenylamine-2-carboxylic acid ( 1 ) . T h e CAS R e g i s t r y N u m b e r is 530 - 7 8 - 9 . 1. 2. F o r m u l a , M o l e c u l a r W ei ght COOH

4JF3 C

H F NO2 1 4 10 3

M o l e c u l a r Weight:

281. 2 4

1. 3 . E l e m e n t a l C o m p o s i t i o n C 59. 79%; €3 3 . 58%; F 20. 27%; N 4. 98%; 0 11. 3 2 % 1. 4. A p p e a r a n c e , C o l o r , O d o r , Taste Pale y e l l o w n e e d l e s , p r a c t i c a l l y o d o r l e s s w i t h a slight bitter t a s t e , 2 . P HYS IC AL P R O P E R T I E S

2 . 1. I n f r a r e d S p e c t r u m T h e IR s p e c t r u m of f l u f e n a m i c a c i d s h o w n in Fig u r e 1 w a s obt ai ned w i t h a B e c k m a n M y c r o l a b 620MX s p e c t r o p h o t o m e t e r i n a K B r p e l l e t c o n t a i n i n g 0. 4 m g of f l u f e n a m i c a c i d / 1 0 0 m g of K B r . S o m e s p e c t r a l a s s i g n m e n t s a r e g i v e n i n T a b l e I. T h i s s p e c t r u m is f n good a c c o r d a n c e w i t h t h e IR s p e c t r u m (1 8 0 0 - 600 c m - ) r e p o r t e d b y K u h n e r t - B r a n d s t ' a t t e r e t al. ( 2 6 ) f o r the p o l y m o r p h i c m o d i f i c a t i o n I11 of f l u f e n a mic a c i d (see S e c t i o n 2. 8).

2. 2. N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r u m T h e 1 H NMR s p e c t r u m of f l u f e n a m i c a c i d is p r e -

92

80

70

50

30

10

10

C 1

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

316

TABLE I

IR Spectral Assignments of Flufenamic Acid Wavenumber, c m - '

Vibration Mode

1670 1612, 1592, 1535 1190, 1125 803, 768, 758, 703

C=O stretching aromatic C=C stretching CF3 a s y m m . s t r e t c h i n g C-H out of plane bending

sented in F i g u r e 2. The s p e c t r u m was r e c o r d e d on a V a r i a n E M - 3 6 0 60 MIlz s p e c t r o m e t e r using a CDC13 solution containing tetramethylsilane a s an i n t e r n a l standard. The s p e c t r a l assignments are given in Table 11, and a r e in accordance with the NMR s p e c t r u m of etofenamate, which is the 2-(2-hydroxyethoxy)ethyl e s t e r of flufenamic acid, reported by Boltze and Kreisfeld (2) a s r e g a r d s the a r o matic protons of the benzene rings. TABLE I1 NMR S p e c t r a l Assignments of Flufenamic Acid

Protons a b C

Chemical Shift, d

6.60-6.96 7.05-7. 53 8. 05

Multiplicity multiple t multiple t doublet

2. 3 . Ultraviolet Spectrum The UV s p e c t r u m of flufenamic acid ( F i g u r e 3 ) w a s scanned f r o m 400 t o 210 nm on a C a r y 219 s p e c t r o -

EN0 OF SWEEP

START OF SWEEP

Fig. 2.

NMR Spectrum of Flufenamic Acid

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

318

F i g . 3.

U l t r a v i o l e t Spectrum of Flufenamic Acid

FLUFENAMIC ACID

319

p h o t o m e t e r , u s i n g a solution of 9. 28 p g of f l u f e n a m i c a c i d / m l of 95% ethanol. T h e a b s o r p t i o n d a t a a r e l i s t e d in Table 111. T A B L E I11 UV Absorption Data of F l u f e n a m i c Acid in 95% ethanol solution

L max'

nm

1c m

4. 32

219 238 2 88 337

745

s houlde r

-

4. 2 2 3. 8 8

5 90 269

The s p e c t r a l f e a t u r e s observed are substantially in a c c o r d a n c e with t h o s e p r e v i o u s l y d e s c r i b e d in the l i t e r a t u r e (3, 4, 5, 6, 7, 8 ) . T h e log & vs. wavelength plot of a m e t h a nolic solution of flufenamic a c i d w a s r e p o r t e d by U n t e r halt ( 3 ) , w h e r e a s Ikeda e t al. ( 5 ) p r e s e n t e d the s p e c t r u m of the d r u g in 0. 1 M phosphate buffer (pH 7. 0 ) . D e l l e t al. (8) r e p o r t e d Amax v a l u e s of flufenamic a c i d and i t s phenolic m e t a b o l i t e s in n e u t r a l , a c i d i c and a l k a l i n e m e thanol. In T a b l e l V are listed s o m e absorption data rep o r t e d in the l i t e r a t u r e . ~~

T A B L E IV UV Absorption D a t a of F l u f e n a m i c Acid 1q" Solvent Amax. n m log E Ref. cm

-

2 88 340.5

4.22 3. 8 9

590 276

3

0 . 0 1 N NaOH

289

4.10

448

3

0 . 1 NNaOH

288

4. 1 8

545

4

0 . 01 N HC1

287 345

4. 2 2 3. 94

5 90 310

3

Methanol

(in CH30H)

320

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

2. 4. M a s s S p e c t r u m T h e mass s p e c t r u m of f l u f e n a m i c a c i d is s h o w n i n F i g u r e 4. T h e s p e c t r u m w a s r u n o n a H e w l e t t - P a c k a r d Mod. 5982A s p e c t r o m e t e r w i t h a n i o n i z i n g e n e r g y of 70 e V , i n t e r f a c e d with a H e w l e t t - P a c k a r d Mod. 5934A d a t a h a n d l i n g s y s t e m . T h e c o m p u t e r c a l c u l a t e d i o n masses a n d compared their peak intensities to the b a s e peak (m/e = 265). T h i s i n f o r m a t i o n w a s t h e n a u t o m a t i c a l l y p l o t t e d a s a series of l i n e s w h o s e h e i g h t s a r e p r o p o r t i o n a l t o t h e peak intensities. T h e molecular ion peak w a s observed a t m / e = 281. S o m e c h a r a c t e r i s t i c p e a k s o b s e r v e d a r e l i s t e d in Table V . T h e f r a g m e n t a t i o n p a t t e r n o b s e r v e d is TABLE V M a s s S D e c t r u m of F l u f e n a m i c A c i d Mass (m/e)

Species

281 263 235 216 166

M+ M+-H20 263-CO 235-F 216-CF2

A b u n d a n c e 01, 44. 8 100.0 22. 8 12. 3 14. 9

c o n s i s t e n t with mass s p e c t r a l d a t a p u b l i s h e d b y B o l t z e a n d K r e i s f e l d ( 2 ) f o r e t o f e n a m a t e a n d C o t e l l e s s a et al. ( 43 ) for t h e m e t h y l e s t e r of f l u f e n a m i c a c i d . 2. 5 . F l u o r e s c e n c e S p e c t r u m F l u f e n a m i c a c i d s h o w s n a t i v e f l u o r e s c e n c e i n some o r g a n i c s o l v e n t s , e . g. d i o x a n a n d c h l o r o f o r m , w h e r e a s i n e t h a n o l f l u o r e s c e n c e is t o o w e a k t o be a n a l i t i c a l l y u s e f u l (8. 9). M i l l e r et al. (10) r e p o r t e d t h a t f l u f e n a m i c a c i d s h o w e d n o s i g n i f i c a n t f l u o r e s c e n c e at room t e m p e r a t u r e i n a c i d i c , n e u t r a l or a l k a l i n e e t h a n o l s o l u t i o n , b u t w a s s t r o n g l y f l u o r e s c e n t at l o w t e m p e r a t u r e ( 7 7 0 K ) , p r e s u m a b l y b e c a u s e of t h e v i r t u a l a b o l i t i o n of b i m o l e c u l a r q u e n c h i n g i n t h e latter c o n d i t i o n s . D e l l a n d K u t s c h b a c h (11) i n v e s t i g a t e d t h e i n f l u e n c e on f l u o r e s c e n c e i n t e n s i t y of t h e s o l v e n t a n d t h e a d d i t i o n of a h a l o g e n o a c e t i c a c i d , a s

00.

80., 60,, 40., E!O#, W

c!

0,

I

F i g . 4.

Mass Spectrum of F l u f e n a m i c A c i d

322

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

well a s the influence of a t r e a t m e n t with a l k a l i a n d / o r a n oxidant. T o obtain a f l u o r e s c e n t solution s o l v e n t s with a d i e l e c t r i c c o n s t a n t = 0 a r e r e q u i r e d , and f l u o r e s c e n c e i n t e n s i t y is s t r o n g l y i n c r e a s e d by t h e addition of a halogen o a c e t i c a c i d with a pKa value < 1 (e. g. t r i c h l o r o a c e t i c a c i d ) . T h e s e findings a r e the b a s i s f o r t h e m o s t c o m m o n m e t h o d of f l u o r o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c a c i d (see Section 5. 5). Dell e t al. (8) published the f l u o r e s c e n c e s p e c t r u m of flufenamic a c i d in a C C 1 4 / t r i c h l o r o a c e t i c a c i d solution: i n t h i s m e d i u m flufenamic a c i d s h o w s the e x c i t a t i o n m a x i m u m a t 372 n m with a s u b s i d i a r y m a x i m u m a t 296 n m , and t h e e m i s s i o n m a x i m u m a t 445 n m . T h e r e is a l i n e a r r e l a t i o n s h i p between the f l u o r e s c e n c e i n t e n s i t y and the c o n c e n t r a t i o n of f l u f e n a m i c a c i d u p t o 1 0 p g / m l . S o m e f l u o r e s c e n c e d a t a r e p o r t e d in the l i t e r a t u r e f o r flufenamic a c i d a r e l i s t e d in T a b l e VI. T A B L E VI F l u o r e s c e n c e Maxima of F l u f e n a m i c Acid in v a ri o u s solvents Solvent Methanol Ethanola Dioxan Chloroform CCl,/TCA

Excitation maximum, n m

Emission m a x i m u m , nm

305 335 3 34 340 372 (296)b

400 510(420)b 410 430 445

Ref.

8 10 9 9 8

a : a t 77OK. b : subsidiary maximum.

2. 6. Melting r a n g e In T a b l e VII a r e l i s t e d s o m e m e l t i n g point v a l u e s r e p o r t e d by v a r i o u s a u t h o r s f o r flufenamic a c i d , T h e d i s c r e p a n c i e s a m o n g t h e s e v a l u e s a r e due t o t h e d i f f e r e n t p o l y m o r p h i c m o d i f i c a t i o n s which c a n be p r e s e n t i n c o m m e r c i a l p r o d u c t s (see Section 2 . 8).

323

FLUFENAMIC ACID

TABLE VII M e l t i n g R a n g e of F l u f e n a m i c A c i d M. P . , OC

125 1 2 4 - 125, with r e s o l i d i f i c a tion a n d r e m e l t i n g a t 134136 127-128 132-133 133 1 3 3 - 134 1 3 3 -1 3 4 o r 1 25- 126, r e s o l id ify in g a n d r e m e l t i n g at 1 3 4 -1 3 6 134. 3 1 3 4 - 136

Crystallization solvent

Ref.

50% e t h a n o l

12

ligroine NR cyclohexane NR NR

13 3 14,15 16 2

NR ethanol cyclohexane

17 18 19

NR : not r e p o r t e d

2 . 7. S o lu b i l i t y, P a r t i t i o n Coef fi ci e n t Flufenamic acid was reported to be soluble a t r o o m t e m p e r a t u r e in m e t h a n o l , e t h a n o l , d i e t h y l e t h e r , c h l o r o f o r m , a c e t o n e , D M F , and p e a n u t oil ( 3 , 4 , 1 3 ) . T h e s o l u b i l i t y in w a t e r a t 22OC is gi ven b y R o l t z e a n d K r e i s f e ld ( 2 ) a s 0 . 0067 mg/ml. In T a b l e VIII a r e l i s t e d s o m e v a l u e s of s o l u b i l i t y in w a t e r a t v a r i o u s p H v a l u e s . A s t u d y b y G h a n e m et al. ( 2 1 ) i n d i c a t e s t h a t t h e s o l u b i l i t y of f l u f e n a m i c a c i d is i n c r e a s e d b y n o n i o n i c s u r f a c t a n t s , u r e a a n d s o d i u m citrate. T h e e f f i c i e n c y of t h e s u r f a c t a n t s t o w a r d s s o l u b i l i z a t i o n is i n t h i s o r d e r : T w e e n 80 > R r i j 9 9 > T w e e n 40 > M y r j 53. T h e e f f e c t of u r e a , a m i d o p y r i n e , p h e n a z o n e a n d p a r a c e t a m o l on t h e s o l u b i l i t y of f l u f e n a m i c a c i d a n d o t h e r a n t i r h e u m a t i c d r u g s w a s s t u d i e d b y D a a b i s e t al. ( 2 2 ) . 1- O c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t w a s e s t i m a t e d b y Dunn (2 3 ) t a k i n g a d v a n t a g e of t h e a d d i t i v e - c o n s t i t u t i v e n a -

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

324

TABLE VIII Solubility of F l u f e n a m i c Acid in Water a t v a r i o u s pH V a l u e s PH

Solubility , mg/ml

Ref,

3 7 7 8

0. 003a 1. 8a 1 4. oa

13 13 20 13

a : a t 37OC t u r e of l o g P, as follows: = log 'anthranilic =

1. 21

acid

+ 2 . 60 + 1 . 0 7

=

+

nphenyl

+

n3-cF3 =

4. 88

T e r a d a e t al. ( 2 4 ) have d e t e r m i n e d the t r u e p a r t i t i o n coefficient, P, a n d the a p p a r e n t p a r t i t i o n coefficient, P', of f l u f e n a m i c acid. P w a s m e a s u r e d by e q u i l i b r a t i n g a 1-octanol solution of the d r u g , the i n i t i a l c o n c e n t r a t i o n , C,, of which w a s 1 0 - 3 - 1 0 - 2 m o l / l , with 0. 01 N HC1: u n d e r t h i s condition, d r u g m o l e c u l e s e x i s t e x c l u s i v e l y a s the unionized f o r m . A f t e r e q u i l i b r i u m w a s a t t a i n e d , c o n c e n t r a t i o n i n the a q u e o u s p h a s e , Cw, was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y and P w a s c a l c u l a t e d b y the equation: P = C,/Cw, s i n c e t h e c o n c e n t r a t i o n change in t h e o r g a nic p h a s e c a n b e n e g l e c t e d f o r s u c h a highly hydrophobic compound, obtaining l o g P = 5 . 62. PI w a s d e t e r m i n e d with the 1- o c t a n o l / p h o s p h a t e b u f f e r (pH 8. 0) s y s t e m , u s i n g the equation:

w h e r e C a n d V a r e t h e e q u i l i b r i u m c o n c e n t r a t i o n and v o l u m e of a q u e o u s ( s u b s c r i p t w) and o r g a n i c ( s u b s c r i p t 0 ) p h a s e s , r e s p e c t i v e l y . Ci is t h e i n i t i a l c o n c e n t r a t i o n i n the a q u e o u s p h a s e . L o g PI w a s found t o b e 1 . 7 4 .

FLUFENAMIC ACID

325

Lombardino et al. (25) have a l s o determined the p a r t i tion coefficient of flufenamic acid with some 1-octanol/ buffer s y s t e m s . 2. 8. Crystal P r o p e r t i e s , Polymorphism Flufenamic acid can exist a s s e v e r a l crystalline modifications. Kuhnert-Brandstatter et al. have d e s c r i bed five different modifications and have reported t h e i r melting points and infrared s p e c t r a , a s well a s the t h e r mogram obtained by differential scanning c a l o r i m e t r y f o r the f i r s t four f o r m s (16, 26). According to K r c flufenamic acid can exist a s at l e a s t seven crystalline modifications with different melting points (27). K r c has reported the f r e e energy vs. t e m p e r a t u r e plot of seven crystalline f o r m s of flufenamic acid. Modifications I, I1 and I11 were described in detail in t e r m s of c r y s t a l morphology, optical p r o p e r t i e s , X - r a y diffraction powder data and infrared s p e c t r a . Other authors a l s o studied the polymorphism of flufenamic acid. Galdecki et al. (28) investigated the c r y s t a l l i zation of the d r u g f r o m boiling solvents, B u r g e r and Ramberger ( 2 9 ) examined the applicability of s o m e t h e r modynamic r u l e s to the polymorphic s y s t e m of flufenamic acid. These r u l e s c o r r e l a t e the heats of transition o r fusion, IR s p e c t r a and densities of the modifications with t h e i r stability behavior. In this study flufenamic acid w a s investigated mainly by quantitative DSC and qualitative s o lubility determination (by thermomicroscopy) a s well a s by IR spectroscopy to differentiate eight crystalline modifications (Table IX). It w a s pointed out ( 2 9 ) that modifications I, I1 and I11 investigated by the various authors a r e identical, whereas modification IV studied by Kuhnert ( 2 6 ) coincides with modification V by K r c ( 2 7 ) , who did Since the l a t t e r not describe Kuhnert's modification V. f o r m had the lowest melting point of all eight modifications, it was indicated by B u r g e r and R a m b e r g e r a s modification VIII. F r o m the practical point of view, modifications I and I11 a r e the most important, because they can be present in the c o m m e r c i a l product. The transition point of these two f o r m s is a t 42°C: modification I11 i s the stable f o r m

326

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

at room t e m p e r a t u r e ( b e l o w 42" C) , w h e r e a s m o d i f i c a t i o n I is t h e s t a b l e form a b o v e 42OC ( 2 7 ) . M o d i f i c a t i o n I11 w a s o b t a i n e d by B u r g e r a n d R a m b e r g e r by s t i r r i n g f o r 1 2 h o u r s a t 2OoC a x y l e n e s u s p e n s i o n of a commercial p r o d u c t f o r m e d by m o d i f i c a t i o n I ( 2 9 ) . T A B L E IX M e l t i n g P o i n t s of c r y s t a l l i n e M o d i f i c a t i o n s of F l u f e n a m i c A c i d Mo d ific a t i on

M. P . , O C

Ref.

I

133 134

16,26 27, 2 9

I1

128

1 6 , 26, 27, 2 9

I11

125 126 126. 5

16,26 29 27

IV

124

27

Va

122 122.5

1 6 , 26, 29 27

VI

120

27

VII

118

27

VIIIb

100-110 108 2 5

26 29

a : T h i s m o d i f i c a t i o n w a s i n d i c a t e d a s IV i n t h e p a p e r s 1 6 a n d 26. b : T h i s modification was indicated as V in the p a p e r s 1 6 a n d 26.

2. 9. D i s s o c i a t i o n C o n s t a n t T h e pKa of f l u f e n a m i c a c i d w a s r e p o r t e d to b e 3 . 9 by A g u i a r a n d F i f e l s k i ( 2 0 ) a n d 4 . 5 by F r e y a n d ElS a y e d (30). Terada et al. ( 2 4 ) h a v e found a v a l u e of 3 . 8 5 u s i n g t h e pH -dependent s o l u b i l i t y m e t h o d ( 3 1 ) ; t h i s value is c o n s i d e r a b l y d i f f e r e n t from t h e c o r r e s p o n d i n g

FLUFENAMIC ACID

327

value obtained by p o t e n t i o m e t r i c t i t r a t i o n i n 5 - 10% a q u e o u s acetone ( 3 2 ) . In T a b l e X a r e l i s t e d s o m e pKa v a l u e s obtained by p o t e n t i o m e t r i c t i t r a t i o n in v a r i o u s a q u e o u s s o l vent s y s t e m s . TABLE X pKa V a l u e s of F l u f e n a m i c Acid obtained bv P o t e n t i o m e t r i c T i t r a t i o n Solvent Water 75% Aqueous methanol 50Vn Aqueous ethanol 80% Aqueous 2-methoxyethanol Dioxane: w a t e r ( 2 : 1)

pKa

Ref.

7. 5 5.75 5. 94

33 3 33

6. 0 6. 8

33 25

3 . SYNTHESIS Wilkinson and F i n a r ( 1 2 ) f i r s t s y n t h e s i z e d flufenamic a c i d by r e a c t i n g o-iodobenzoic a c i d with m - t r i f l u o r o m e thylaniline in p o t a s s i u m c a r b o n a t e a q u e o u s solution, i n t h e p r e s e n c e of c o p p e r b r o n z e . T h e c r u d e p r o d u c t w a s p u r i fied via i t s a m m o n i u m s a l t . Moffett and A s p e r g r e n (19) p r e p a r e d f l u f e n a m i c a c i d s t a r t i n g f r o m o - c h l o r o b e n z o i c a c i d which w a s r e a c t e d with m-trifluoromethylaniline i n 85070 aqueous p o t a s s i u m hydroxide and a m y l alcohol with c o p p e r p o w d e r . Some patented synthetic m e t h o d s follow the l a t t e r s c h e m e , as i l l u s t r a t e d in F i g u r e 5.

Figure 5 Synthesis of F l u f e n a m i c Acid F l u f e n a m i c a c i d w a s a l s o obtained via the r e a c t i o n b e t ween o-iodobenzoic a c i d and m-trifluoromethylphenylhydroxylamine ( 3 4 ) . Another method involves the r e a c t i o n of

328

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

m e t h y l o - c h l o r o b e n z o a t e with N-(3-trifluoromethylphenyl)f o r m a m i d e (35). F l u f e n a m i c a c i d w a s a l s o p r e p a r e d by photolysis of the c o r r e s p o n d i n g b e n z o t r i a z i n o n e ( 3 6 ) . 4. DRUG METABOLISM AND PHARMACOKINETICS 4. 1. M e t a b o l i s m T h e m e t a b o l i c t r a n s f o r m a t i o n s which f l u f e n a m i c a c i d u n d e r g o e s in m a n and a n i m a l s a r e d e p i c t e d in F i g u r e 6 , a c c o r d i n g t o Glazko ( 3 7 ) and O b e r e t al. (38), which d e m o n s t r a t e d that t h e d r u g is e x c r e t e d m a i n l y i n f o r m of i t s m e t a b o l i t e s . T h e i r s t u d i e s w e r e c a r r i e d out by t r a c e r m e t h o d s u s i n g [14C] - c a r b o x y l - l a b e l l e d f l u f e n a m i c acid. T h e 4'-hydroxy- and 5 - h y d r o x y - d e r i v a t i v e and f l u f e n a m i c a c i d itself a r e e l i m i n a t e d in u r i n e chiefly i n conjugated f o r m , w h e r e a s t h e 4', 5 - d i h y d r o x y - d e r i v a t i v e is not conjugated. A l l f o u r compounds h a v e b e e n found i n t h e s t o o l i n unconjugated f o r m . 4'-Hydroxy- and 5-hydroxyflufenamic a c i d s w e r e s y n t h e s i z e d by Bowman et al. (39). 4. 2. Absorption and E x c r e t i o n 4. 21.

In A n i m a l s T h e r a t e of p e r m e a t i o n of f l u f e n a m i c a c i d t h r o u g h the gut m e m b r a n e and the amount of d r u g a b s o r bed w e r e studied i n v i t r o on s e g m e n t s of t h e small i n t e s t i n e of golden h a m s t e r s ( 2 0 ) . T h e p e r m e a t i o n of flufenam i c a c i d w a s pH-dependent, a c c o r d i n g t o t h e postulation t h a t i t is only the unionized m o l e c u l e of t h e d r u g t h a t pass e s through a cell, due t o i t s lipoid solubility. T h i s s t u d y showed t h a t a t pH 2. 5 the p e r m e a t i o n of f l u f e n a m i c acid, which is a p p r o x i m a t e l y 96% unionized, is 20 t i m e s f a s t e r t h a n a t pH 7. 2, at which only 0. 05% of t h e d r u g is i n the unionized f o r m . T h e a b s o r p t i o n and t h e u r i n a r y e x c r e t i o n of f l u f e n a m i c a c i d w a s s t u d i e d i n r a b b i t s following both c u t a n e o u s and o r a l application ( 4 0 ) of t h e same d o s e ( 3 0 m g / k g ) . A f t e r 4 8 h o u r s from cutaneous application, 5. 9% of t h e applied d o s e w a s found i n the u r i n e ; t h e blood level of flufenam i c a c i d r e m a i n e d c o n s t a n t o v e r the f i r s t s i x h o u r s at about 3 pg/ml, o v e r c o m i n g t h e blood l e v e l obtained o r a l l y a s f r o m the 4th hour.

m

f 4 R Erc

m

u.

0

r

I

R

q Erc

&

0 I

u3

/ \ 4

0 c k I V

0

I

CD

a,

lil

.rl~

R

c

a,

I

rcl

Erc

rl

0

+I

E

(I)

0

d .rl

Y

lu

e

3 30

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

Rosenberg and Bates ( 4 1 ) compared the blood concentrations produced in r a t s following o r a l administration of f l u fenamic acid alone o r together with cholestyramine. F l u fenamic acid strongly bound to the r e s i n , s o that a 6 0 70% d e c r e a s e in both the r a t e and the extent of absorption of the d r u g was observed: following an o r a l dose of 5 0 m g / k g a peak plasma concentration of 8 9 . 2 p g / m l w a s reached at 1 hour, whereas when cholestyramine and the d r u g were coadministered the peak l e v e l dropped to 3 2 . 3 F u r t h e r studies on pharmacokinetics of flufenapg/ml. m i c acid in the r a t a r e reported. F r e y and El-Sayed ( 3 0 ) determined the flufenamic acid concentrations in s e r u m and g a s t r i c mucosa a f t e r o r a l and subcutaneous administration. Lin et al. ( 4 2 ) determined p l a s m a l e v e l s a f t e r intravenous administration, Cotellessa e t al. ( 4 3 ) d e t e r mined plasma and u t e r u s levels a f t e r intravenous and o r a l administration. A s r e g a r d s the excretion, Glazko ( 3 7 ) showed that dogs eliminated only 2-770 of an o r a l dose in the urine and 537 9 % in the f e c e s , w h e r e a s the corresponding values f o r monkeys were 45-8070 and 12-2170, respectively, S i m i l a r r e s u l t s were obtained by Ober et al. ( 3 8 ) in e x p e r i m e n t s with dogs. Lombardino et al. ( 2 5 ) r e p o r t e d that the s e r u m half life a f t e r o r a l administration of the aluminium salt of flufenamic acid w a s 3 h o u r s f o r rats and dogs, and 4 hours f o r rabbits.

4 . 2 2 . In Humans In addition to the r e s u l t s obtained in r a b bits ( 4 0 ) , P a n s e e t al. have a l s o studied the absorption of flufenamic acid through the human skin ( 4 4 ) . Glazko ( 3 7 ) reported that n e a r l y 100% of an o r a l dose of flufenamic acid w a s absorbed; the r e n a l elimination of the drug and i t s metabolites was 5170, of which only 2 . 6% w a s unaltered drug. Such r e s u l t s were m o r e r e c e n t l y confirmed by Dell e t al. ( 4 5 , 4 6 ) with two different methods for the determination of all fluorine-containing c o m pounds a s a group in the urine, which showed that the r e nal elimination of flufenamic acid and i t s metabolites w a s 4 9 . 470 within t h r e e days a f t e r o r a l administration. The peak p l a s m a level w a s reached a f t e r two hours, and the p l a s m a elimination half life w a s found t o be approximate-

FLUFENAMIC ACID

331

l y 3 h o u r s . D e l l e t al. ( 4 5 ) a l s o r e p o r t e d t h a t 3. 670 of a n o r a l d o s e of flufenamic a c i d was e x c r e t e d unconjugated into the u r i n e within 6 d a y s : however, f e m a l e s u b j e c t s e l i m i n a t e d only 1. 9% and the m a l e o n e s 5. 370. On the c o n t r a r y , no difference w a s o b s e r v e d between m e n and women in the t o t a l amount of all m e t a b o l i t e s e x c r e t e d by the r e n a l route. Another study on t h e sex-dependence of the r e n a l e x c r e t i o n of flufenamic a c i d and o t h e r f e n a m a t e s in m a n and a n i m a l s was r e p o r t e d by L o r e n z a n d D e l l (47). T h e bioavailability of o r a l p h a r m a c e u t i c a l f o r m u l a t i o n s of flufenamic a c i d w a s investigated by A r i a s a n d Cadorniga ( 4 8 ) and Angelucci e t al. (49). 4. 3 . P r o t e i n binding T h e bovine and human s e r u m a l b u m i n (BSA and HSA, r e s p e c t i v e l y ) binding affinity of flufenamic a c i d w a s investigated by Chignell b y c i r c u l a r d i c h r o i s m s t u d i e s (50-52). T h e r o l e of hydrophobicity f o r the binding affinit y w a s investigated by Dunn on HSA (23) and by T e r a d a e t al. on BSA (24). T h e hydrophobicity a s well a s the withd r a w i n g ability of the - C F 3 substituent c o n t r i b u t e significantly t o the binding affinity, which w a s d e t e r m i n e d f o r BSA by m e a s u r i n g the ability of flufenamic a c i d t o d i s p l a c e 2 - (4' - hydroxypheny1azo)benzoic a c i d c o m p e t i t i v e l y u n d e r conditions of pH 7 . 0 and 25OC (24). T h e binding constant, K, w a s d e t e r m i n e d : the value obtained f o r BSA by T e r a d a e t a l . , 6 . 5 l~o 5 1. m o l - l , s e e m s t o c o n f o r m t o a v a lue, 1. 3 x l o 6 1. m o l - l , obtained by Chignell with HSA at pH 7 . 4 (51). T h e i n t e r a c t i o n between BSA and s e v e r a l c a t i o n i c and anionic d r u g s including flufenamic a c i d w a s studied by B l a n c h a r d e t al. (53) u s i n g the e l e c t r o n s p i n r e s o n a n c e s p i n labeling technique. T h e binding of flufenamic a c i d t o HSA w a s s t u d i e d by O t a g i r i e t al. ( 5 4 ) v i a m i c r o c a l o r i m e t r i c i n v e s t i g a t i o n s . T h e h e a t flux g e n e r a t e d by the binding is p r o p o r t i o n a l t o the amount of the d r u g bound t o the p r o t e i n . If only one binding s i t e on t h e d r u g m o l e c u l e c o n t r i b u t e s t o the h e a t flux, then the d a t a can r e a d i l y be i n t e r p r e t e d i n t e r m s of the binding constant, AG, AH, and A S f o r binding t o that site. If m a n y s i t e s a r e involved having d i f f e r e n t e n t h a l -

332

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

pies of binding, then unambiguous interpretation of the data may be impossible. T h i s is the c a s e of flufenamic acid, which h a s been reported t o have t h r e e v e r y high affinity s i t e s f o r HSA and other s i t e s of lower affinity(51). Sudlow et al. (55, 56) have c h a r a c t e r i z e d two distinct binding s i t e s ( I and 11) f o r anionic d r u g s on HSA by the u s e of fluorescent probes. Flufenamic acid binds s e l e c t i vely to s i t e 11, a s well a s ibuprofen, flurbiprofen and ethacrynic acid , whereas phenylbutazone and warfarin bind to s i t e I. Drugs which bind t o s i t e I1 a r e all a r o m a t i c carboxylic acids, which would be l a r g e l y ionized a t physiological pH. Kaneo et al. (57) examined the binding t o BSA of s i x nonsteroidal antiinflammatory d r u g s including flufenamic acid by the u s e of dialysis a t pH 7 . 4 and 37OC. It was found that flufenamic acid strongly binds t o BSA and the f r e e fraction of t h i s d r u g e x i s t s within 1% over the t h e r a peutic range. 5. METHODS OF ANALYSIS 5. 1. Identification T e s t s Flufenamic acid can be identified by virtue of i t s U V , IR, N M R , m a s s and fluorescence s p e c t r a (see Section 2). Various chromatographic methods a r e a l s o s u i table f o r purposes of identification ( s e e Section 5 . 7 ) . Devaux e t al. (58) described two color r e a c t i o n s and one fluorescent reaction. The color r e a c t i o n s a r e due t o the diphenylamine s t r u c t u r e , w h e r e a s the fluorescent r e a ction was explained by the formation of substituted a c r i dones ( s e e Section 5.5). T h e s e reactions can be c a r r i e d out a s follows: a ) Flufenamic acid (at l e a s t 1 m g ) and about 0. 5 g of oxalic acid a r e heated into an oil bath at 180-200°C 4-5 minutes. After cooling the r e s i d u e is dissolved in 95% ethanol t o obtain a stable, intense blue color. The a b s o r ption maximum is at 585-590 nm. b ) Flufenamic acid ( a t l e a s t 100 p g ) is added in a mixt u r e of 1 m l CH3COOH:H2S04 (d.1.83)(98:2), 5 rnl CH3CO0H:HCl (d. 1.18)(50:50), and 1 m l 0. 10% aqueous levulose. The mixture is heated a t 100°C 25 minutes t o ob-

FLUFENAMIC ACID

333

tain a violet color. The absorption maximum is at 597nm. c ) Flufenamic acid is dissolved in conc. H2SO4 and heated 10 minutes at 100°C: the solution exhibits an intense green fluorescence when excited by white light, and blue when excited by UV light. 5. 2. T i t r i m e t r i c Analysis Flufenamic acid can be titrated in acetone with 0 . 1 N aqueous potassium hydroxide in the p r e s e n c e of phenolphtaleine (3). A nonaqueous t i t r i m e t r i c method w a s described by Walash and Rizk (59), which used 0 . 1 N s o dium methoxide as the t i t r a n t and DMF o r tetramethylu r e a as the solvent, m e a s u r i n g the end point e i t h e r with a thymol blue indicator o r potentiometrically. Various potentiometric methods in aqueous solvent s y s t e m s a r e cited in Section 2. 9. 5. 3. Colorimetric Analysis Flufenamic acid and i t s metabolites w e r e d e t e r mined a s a group colorimetrically in urine a f t e r alkaline hydrolysis and fusion with sodium peroxide: fluorine was then distilled a s H2SiF6 in the p r e s e n c e of H2S04 and Si02. A. solution of alizarine-3-methylamino-N, N-diacetato-cerium(II1) was added to the distillate to obtain a col o r reaction, and a c o l o r i m e t r i c determination w a s effected at 617 nm. Measurements in the range h l p,g a r e possible (46). The color reactions cited in Section 5. 1 can a l s o be used for quantitation. 5.4. Spectrophotometric Analysis The f i r s t UV spectrophotometric method f o r fenam a t e s analysis w a s described by Carey (60). The W absorbance of flufenamic acid ( s e e Section 2. 3 ) e i t h e r in methanol a t 288 nm (3,40,44) o r in 0. 1 N NaOH at 287290 nm (4, 30,411 can be used f o r quantitative analysis. Beltagy (61) described a spectrophotometric method f o r the determination of s e v e r a l acidic d r u g s including flufenamic acid which w a s determined obtaining the ion-pair association complex of the d r u g and safranine in a pH 7. 4 buffer, then extracting the complex with chloroform and measuring the absorbance of the e x t r a c t .

334

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

5. 5. F l u o r o m e t r i c A n a l y s i s Mehta and Schulman ( 9 ) a f f i r m e d t h a t t h e native f l u o r e s c e n c e exhibited by f l u f e n a m i c a c i d i n o r g a n i c s o l v e n t s (see Section 2. 5) could b e u s e f u l f o r i t s d e t e c t i o n and d e t e r m i n a t i o n , N e v e r t h e l e s s the m e t h o d m o s t corn monly u s e d involves the f l u o r o m e t r i c d e t e r m i n a t i o n of fluf e n a m i c a c i d i n c a r b o n t e t r a c h l o r i d e a f t e r addition of a CC14 solution of t r i c h l o r o a c e t i c a c i d which s t r o n g l y i n c r e ases the f l u o r e s c e n c e i n t e n s i t y (11). T h e f l u o r e s c e n c e m a x i m a u n d e r t h i s condition a r e r e p o r t e d i n Section 2. 5. T h i s method w a s u s e d by a n u m b e r of a u t h o r s t o a s s a y flufenamic a c i d i n body f l u i d s and t i s s u e s (8, 37, 40, 49, 62, 63, 64). T h e r e a c t i o n of f l u f e n a m i c a c i d with f o r m a l d e h y d e gives 1-(m-trifluoromethylphenyl)-4-oxo-1,2 - d i h y d r o - 3, 1- b e n z o xazine (4), which is s u i t a b l e f o r f l u o r o m e t r i c d e t e r m i n a tion of the p a r e n t d r u g (63). T h e r e a c t i o n s c h e m e is d e picted in F i g u r e 7. T h e m e t h a n o l i c s o l u t i o n of t h e b e n z o xazine d e r i v a t i v e s h o w s two e x c i t a t i o n m a x i m a at 278 and COOH

NH I

0

HCHO

Figure 7 Reaction of F l u f e n a m i c Acid with F o r m a l d e h y d e 342 n m and a n e m i s s i o n m a x i m u m a t 440-450 n m ( 4 ) . T h e c o r r e s p o n d i n g v a l u e s r e p o r t e d by D e l l e t al. ( 6 3 ) a r e 346 and 458 nm. Another fluorogenic r e a c t i o n of f l u f e n a m i c a c i d , a l r e a d y c i t e d i n Section 5. 1, w a s s t u d i e d by D e l l and K a m p ( 4 ) . F l u f e n a m i c a c i d w a s h e a t e d with c o n c e n t r a t e d s u l f u r i c a c i d to give a m i x t u r e of two i s o m e r i c a c r i d o n e s , I and 11, as i l l u s t r a t e d i n F i g u r e 8. T h e f l u o r e s c e n c e f e a t u r e s of t h e s e compounds, which had b e e n p r e v i o u s l y s y n t h e s i zed by Wilkinson and F i n a r (12), a r e v e r y s i m i l a r s o that

FLUFENAMIC ACID

335

Reaction of F l u f e n a m i c Acid with conc. H2S04 the quantitative d e t e r m i n a t i o n c a n b e m a d e on t h e mixtur e . In T a b l e XI a r e l i s t e d the wavelengths of excitation and e m i s s i o n m a x i m a i n n e u t r a l , a c i d i c and a l k a l i n e m e thanol ( 4 ) . T A B L E XI F l u o r e s c e n c e Data of Trifluoromethylacridones Solvent system Methanol Methanol- H C1 Methanol - NaOH

E x c i t a t i o n / e m i s s i o n m a x i m a , n m , of: 4 - C F 3 - a c r i d o n e (I) 2 - C F 3 - a c r i d o n e (11) 400 / 420 400/440 400 / 4 5 5 - 4 6 2

400 / 4 2 1 400/440 400/460

H a t t o r i e t al. ( 6 5 ) p r o p o s e d a f l u o r o m e t r i c method which involves the t r e a t m e n t of a n ethanolic solution of flufenamic a c i d with 0. 5% A1C13 solution i n a b s o l u t e e t h a nol t o obtain a n a l u m i n i u m c h e l a t e , which f l u o r e s c e s a t 440 n m following activation a t 358 n m . T h e m a x i m u m f l u o r o m e t r i c s e n s i t i v i t y of flufenamic a c i d c l a i m e d f o r t h i s method is 4 n g / m l .

5 . 6. I n d i r e c t Atomic Absorption A n a l y s i s It w a s found that flufenamic acid, c o p p e r and 2-(2-hydroxyethyl)pyridine combined i n the r a t i o 1:1:1 t o f o r m a c h e l a t e complex (18). T o obtain t h i s r e s u l t the s a m p l e containing the d r u g w a s t r e a t e d with a r e a g e n t p r e p a r e d adding 9. 0 m l of 0. 1% c u p r i c s u l f a t e solution t o

336

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

1.5 m l of 2-(2-hydroxyethyl)pyridine. The complex w a s then extracted with propyl acetate, and the amount of flufenamic acid p r e s e n t in the s a m p l e was obtained i n d i r e c tly f r o m the amount of copper determined in the organic solvent by atomic absorption analysis. 5. 7. Chromatographic Analysis 5.71. P a p e r Chromatography Flufenamic acid can be detected by p a p e r chromatography (66) using the following solvent s y s t e m s : a ) Methyl isobutyl ketone : f o r m i c acid : w a t e r (10 p a r t s of ketone s a t u r a t e d with 1 p a r t of 470 f o r m i c acid); Rf = 0.95. b ) Chloroform : methanol : f o r m i c acid : w a t e r (a mixtur e of 1 p a r t of methanol and 1 p a r t of 470 f o r m i c acid used t o s a t u r a t e 10 p a r t s of chloroform); Rf = 0. 95. c ) Benzene : methyl ethyl ketone : f o r m i c acid : water ( a mixture of 9 p a r t s of benzene and 1 p a r t of ketone saturated with 1 p a r t of 2’70 f o r m i c acid); Rf = 0. 94. d ) Benzene : f o r m i c acid : water (10 p a r t s of benzene s a t u r a t e d with 1 p a r t of 270 f o r m i c acid); Rf = 0. 91. e ) Methyl ethyl ketone : diethylamine : w a t e r (921:2:77); Rf = 0. 83. f ) Methyl ethyl ketone : acetone : f o r m i c acid : water (40:2:1:6); Rf = 0. 95. Flufenamic acid can be visualized by UV light, or s p r aying the p a p e r with 0.470 p-nitrobenzenediazonium fluoborate solution in 1:2 dioxane:water o r with 2’70 aqueous phosphomolybdic acid solution. Schmollack and Wenzel (67) developed a method f o r the detection and quantitative determination of flufenamic acid using a c h a m b e r p a p e r a n a l y s i s apparatus. Flufenamic acid w a s then determined fluorometrically a f t e r t r e a t m e n t with formaldehyde vapor t o obtain the strongly fluorescent benzoxazine derivative. 5.72. Thin L a y e r Chromatography Several thin l a y e r chromatographic methods have been developed f o r identification and quantitative determination of flufenamic acid. Some d e t a i l s of these methods a r e s u m m a r i z e d in Table XII. It h a s t o point out that in all c a s e s s i l i c a g e l plates w e r e used.

T A B L E XI1 T h i n L a y e r C h r o m a t o g r a p h y of F l u f e n a m i c Acid Solvent S y s t e m

Ref. 3

Cyclohexane:CHC13:CH30H:CH3COOH (60:30:5:5) B e n z e n e : e t h e r : CH3 COOH: CH3 OH ( 120 : 60: 18: 1)

Plates

Rf

D e t e c t ion

S. G. G F

0. 54

W(254nm)

0. 7 8 NR

id. UV following h e a t i n g with HCHO W ( 3 5 6 nm)

3 4

Benzene:C2H50H:CH3COOH (20:2:1)

id. S. G. H F

8

Cyclohexane :e t h y l acetate : CH3 COOH ( 2 0 :30 :2 )

S. G. F 6 0

0.58

8

Cyclohexane: CHC13:CH3COOH (40:50: 10)

S. G . H F

NR

id.

0.43

W

S. G. H F

NR

UV

id.

0. 58

40

id.

S.G. H F F

44

id.

63

id.

U V ; heat. HCHO + U V ; iodine id.

63

B e n z e n e : m e t h a n o l (9: 1)

id.

63

Cyc1ohexane:ethyl acetate (1:1 0 )

id.

0. 23

id.

41

1sopropanol:ammonia:water (20: 1 : 2 )

NR

0. 64

NR

68

To1uene:acetic a c i d ( 9 : l )

S. G. G

0.73

68

To1uene:acetic a c i d (97. 5:2. 5 )

69

Chloroform:methanol(7:3) i n NH3 atm.

S. G . : silica gel.

NR : not r e p o r t e d .

,

0. 23

id.

0. 60

S.G. 60

0. 37

H N 0 2 spray id.

HCHO/HCOOH at

loooc + w

338

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

Unterhalt ( 3 ) h a s identified f l u f e n a m i c a c i d i n a m i x t u r e with o t h e r n o n s t e r o i d a l a n t i i n f l a m m a t o r y d r u g s : the s p o t v i s u a l i z e d by UV light w a s e l u t e d with m e t h a n o l and flufen a m i c a c i d w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 288 n m with a m e a n r e c o v e r y of 88%. Dell and K a m p ( 4 ) a s s a y e d f l u f e n a m i c a c i d in u r i n e and s e r u m : t h e i r method involved a n e t h e r e a l e x t r a c t i o n f r o m t h e biological fluids followed by T L C . T h e p l a t e s w e r e then exposed t o f o r m a l d e h y d e v a p o r ( 2 h o u r s at 8OoC) and t h e UV-fluorescent spot w a s eluted with m e t h a n o l and f l u f e n a m i c a c i d d e t e r m i n e d f l u o r o m e t r i c a l l y . An a l t e r n a t i v e method p r o p o s e d by the s a m e a u t h o r s involved t h e v i s u a lization of flufenamic a c i d by UV light and a t r e a t m e n t of the s c r a p e d off s i l i c a g e l with conc. H2SO4 followed by f l u o r o m e t r i c d e t e r mi nat ion. T h e method d e s c r i b e d by P a n s e e t a l . ( 4 0 ) f o r t h e d e t e r m i n a t i o n of flufenamic a c i d in u r i n e and p l a s m a w a s s i m i l a r , involving e x t r a c t i o n f r o m b i o l o g i c a l m a t e r i a l , T L C and elution of the d r u g , followed by q u a n t i t a t i v e d e termination ei t h e r spectrophotometrically in methanolic solution o r f l u o r o m e t r i c a l l y in a CC14 solution i n the p r e s e n c e of t r i c h l o r o a c e t i c a c i d . T h e l a t t e r m e t h o d w a s a p plied by s e v e r a l a u t h o r s ( a l r e a d y c i t e d in Section 5. 5) f o r t h e d e t e r m i n a t i o n of f l u f e n a m i c a c i d in b i o l o g i c a l fluids and t i s s u e s . P a n s e e t al. ( 4 4 ) d e s c r i b e d a l s o a method f o r t h e d i r e c t d e n s i t o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c a c i d on the s i l i c a gel thin l a y e r s . F l u f e n a m i c a c i d w a s d e t e r m i n e d d i r e c t l y in p l a s m a by G e i s s l e r e t a l . ( 6 9 ) by addition of m e t h a n o l t o p r e c i p i t a t e p r o t e i n s , T L C , t r e a t m e n t of t h e d r i e d p l a t e with f o r m a l dehyde v a p o r in t h e p r e s e n c e of f o r m i c a c i d a t 100°C f o r 45 m i n u t e s t o f o r m t h e benzoxazine d e r i v a t i v e , and d i r e c t f l u o r i m e t r y of t h e plate. Use of f o r m i c a c i d s h o r t e n s t h e r e a c t i o n t i m e and e n h a n c e s t h e f l u o r e s c e n c e i n t e n s i t y and the sensitivity (quantities I 2 ng/spot m a y be detected). A method involving r e v e r s e d - p h a s e thin l a y e r c h r o m a t o g r a p h y w a s r e p o r t e d by B o l t z e and K r e i s f e l d (2), which u s e d s i l i c a gel p l a t e s i m p r e g n a t e d with a 10'70 solution of Dow-Corning 200 i n e t h e r . F l u f e n a m i c a c i d w a s c h r o m a t o g r a p h e d u s i n g buffer : dioxane : a c e t o n e (2: 1 : l ) a s the s o l vent s y s t e m , in which t h e buffer w a s a t pH 5. 2 , 6. 2, and

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7. 2, r e s p e c t i v e l y . In T a b l e XI11 a r e l i s t e d Rf and R M v a l u e s found f o r flufenamic a c i d . T A B L E XI11 Reversed-Dhase T L C of F l u f e n a m i c Acid pH of the buffer 5. 2 6. 2 7. 2

-

Rf

RM= l o g ( l / R f - l )

0. 78 0. 76 0. 69

- 0.55 0.5 - 0. 350 -

5.73. Gas Chromatography Roseboom and Hulshoff ( 7 0 ) h a v e developed a r a p i d and s i m p l e c l e a n - u p and d e r i v a t i z a t i o n p r o c e d u r e that c a n be g e n e r a l l y applied t o the g a s c h r o m a t o g r a p h i c d e t e r m i n a t i o n of a c i d i c d r u g s including f l u f e n a m i c a c i d i n p l a s m a s a m p l e s . T h e d r u g w a s e x t r a c t e d f r o m acidified p l a s m a with c h l o r o f o r m : i s o p r o p a n o l (95:5), which w a s then e v a p o r a t e d . T h e r e s i d u e w a s d i s s o l v e d i n toluene, then the d r u g w a s b a c k - e x t r a c t e d with a small v o l u m e of a methanolic 20% t e t r a m e t h y l a m m o n i u m hydroxide solution (TMAH). T h e solution obtained w a s added t o N , N - d i m e t h y l a c e t a m i d e . A f t e r t r e a t m e n t with n-butyl iodide the d r u g w a s c h r o m a t o g r a p h e d a s i t s n-butyl ester. A gas c h r o m a t o g r a p h equipped with a flame ionization d e t e c t o r w a s u s e d . T h e g l a s s c o l u m n s (150 c m x 2 mm I. D. ) w e r e packed with 3% OV-1, 3% OV-17 o r 3y0 SP-1000, all on 100-120 m e s h C h r o m o s o r b W HP. T h e c a r r i e r gas ( n i t r o g e n ) f l o w - r a t e w a s mantained a t 20 m l / m i n . T h e r e c o v e r y of flufenamic a c i d in the first e x t r a c t i o n s t e p with ch1oroform:isopropanol w a s 69%, but with toluene, which c a n a l s o be u s e d f o r the e x t r a c t i o n f r o m p l a s m a , a r e c o v e r y of 95% w a s achieved. Toluene h a s the advantage that no e v a p o r a t i o n of the e x t r a c t is n e c e s s a r y , and it can b e e x t r a c t e d d i r e c t l y with the TMAH solution. In t h e b a c k - e x t r a c t i o n with TMAH a r e c o v e r y of 62Y0 w a s obtained. T h e r e t e n t i o n t i m e s of the n-butyl e s t e r of flufenam i c a c i d with v a r i o u s s t a t i o n a r y p h a s e s a r e l i s t e d i n Table XIV.

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ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

TABLE XIV G a s ChromatoaraDhic Data f o r Flufenamic Acid (701

Stationary phase 370 ov-1 370OV-17 370 sP-1000

Column temperature, OC

Re tent ion time, sec

2 00 210 230

2 34 271 218

Another g a s chromatographic method f o r quantitative determination of flufenamic acid in p l a s m a w a s reported by Cotellessa et al. (43). Flufenamic acid w a s extracted f r o m plasma with benzene, a f t e r dilution of p l a s m a s a m ple with an equal volume of 0. 25 M acetate buffer (pH 4.35). The benzene e x t r a c t w a s evaporated to d r y n e s s under vacuum. The r e s i d u e w a s dissolved in methanol and methylated with diazomethane. The s a m e procedure w a s a l s o applied t o r a t u t e r u s homogenates. The methylated sample w a s dissolved in a n acetone solution of the i n t e r n a l standard 3-chloro-6-aminobenzophenone. G a s chromatographic analysis w a s c a r r i e d out using a gas chromatograph equipped with a flame ionization detector. The stationary phase w a s 370 OV-17 on Gas-Chrom Q (100-120 m e s h ) packed into a g l a s s column ( 3 m x 2 m m I. D. ). The column t e m p e r a t u r e w a s 23OoC and the c a r r i e r g a s (nitrogen) flow-rate w a s 40 m l / m i n . F I D s e n s i tivity was 1 p g / m l p l a s m a and 5 ,ug/g u t e r u s . A m a s s s p e c t r o m e t e r coupled with the g a s chromatograph was employed to a s c e r t a i n the identity of the m e thyl e s t e r of flufenamic acid with the GC peak. A m a s s s p e c t r u m of the methyl e s t e r is presented. The recover i e s f r o m plasma in the range 10-100 ,ug/ml and f r o m u t e r u s homogenates were 98. 270 and 9070, respectively. A g a s chromatographic method f o r the detection of nons t e r o i d a l antiinflammatory d r u g s including flufenamic acid in urine collected f r o m h o r s e s that had received these compounds orally h a s been developed by Hunt et al. (71). T h i s procedure involves the isolation of the d r u g s f r o m

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341

urine by solvent extraction and on-column methylation of the carboxylic acid group. 5. 74. High P e r f o r m a n c e Liquid Chromatography A method f o r the separation and d e t e r m i n a tion of some nonsteroidal antiinflammatory d r u g s including flufenamic acid w a s described by Dusci and Hackett ( 7 2 ) . This procedure, which can be applied to s e r u m s a m p l e s of s m a l l volume (100 pl), involved the extraction of the drugs with acetonitrile. The e x t r a c t w a s taken to d r y n e s s at 5OoC under a s t r e a m of nitrogen. The r e s i d u e w a s redissolved in 1 0 0 pl of the elution solvent (60% acetonitrile in 45 mM KH2P04 adjusted to pH 3. 0 with H 3 P 0 4 ) . An aliquot of 10-20 pl was injected in a high performance liquid chromatograph equipped with a variable wavelength U V detector. The column (30 c m x 3. 9 m m I. D. ) w a s packed with pBondapak c18. The conditions f o r individual analysis of flufenamic acid were as follows: flow-rate of the elution solvent 2. 0 ml/min, wavelength 282 nm. F o r the separation of flufenamic acid f r o m the mixture of antiinflammatory drugs (flufenamic and mefenamic acid, naproxen , ibuprofen , indomethacin, phenylbutazone, oxyphenbutazone) a flow-rate of 0 . 8 m l / m i n and a wavelength of 225 nm was used: under these conditions the elution t i m e of flufenamic acid w a s 10. 5 min. Using the above -mentioned elution solvent , flufenamic and mefenamic acid were not separated. A modified elution solvent (3570 acetonitrile in 0. 7% NHqC1 buffered to pH 7. 8 with ammonia) allowed to obtain the separation of all the drugs investigated. Using a flow-rate of 1. 0 m l / min, the elution time of flufenamic acid w a s 10. 2 min (mefenamic acid 7. 8 min). The r e c o v e r y of flufenamic acid was 92t370 in a s e r i e s of ten plasma s a m p l e s e x a m i ned, in the range 1. 0-20 p g / m l . Lin et al. ( 4 2 ) have developed a H P L C procedure f o r the determination of flufenamic acid and mefenamic acid in plasma, A single extraction s t e p i s followed by r e v e r sed-phase chromatography. Flufenamic acid and mefenamic acid can be internal standards f o r each other during e i t h e r assay. The extraction of flufenamic acid f r o m a c i dified plasma s a m p l e s (1 m l ) , t o which 4 p g of mefenamic acid had been added, w a s accomplished with carbon t e t r a -

342

ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS

chloride. T h e e x t r a c t w a s e v a p o r a t e d t o d r y n e s s u n d e r a s t r e a m of nitrogen and t h e r e s i d u e w a s r e d i s s o l v e d i n t o 0. 5 m l of methanol, and a n aliquot w a s i n j e c t e d in t h e c h r o m a t o g r a p h , which w a s equipped with a s t a i n l e s s steel column ( 3 0 c m x 4 m m I. D. ) packed with a s t a b l e r e v e r s e d - p h a s e s t a t i o n a r y p h a s e of p o r o u s silica b e a d s c o a t e d with c h e m i c a l l y bonded cyanopropylsilane m o n o l a y e r s . T h e elution solvent w a s w a t e r : a c e t o n i t r i l e : a c e t i c a c i d (60:30: 10). T h e f l o w - r a t e w a s 1 m l / m i n with a n o p e r a t i n g p r e s s u r e of 1000 p s i at r o o m t e m p e r a t u r e . T h e effluent w a s m o n i t o r e d continuously at 254 n m . Under t h e s e conditions flufenamic a c i d had an elution t i m e of 10. 4 min, with a m e a n r e c o v e r y f r o m p l a s m a of 100.7?3.4% in t h e 1-10 p g r a n g e . T h e s e n s i t i v i t y l i m i t w a s 1 p g / m l of p l a s m a . A method f o r t h e d e t e r m i n a t i o n of flufenamic a c i d a n d i t s m a j o r m e t a b o l i t e s , 4 ' - h y d r o x y - and 5-hydroxyflufenam i c a c i d , w a s d e s c r i b e d by Kubo e t al. ( 7 3 ) : t h e a c i d i fied s e r u m w a s e x t r a c t e d with ethyl a c e t a t e . T h e o r g a n i c e x t r a c t was e v a p o r a t e d and the r e s i d u e w a s r e d i s s o l v e d i n ethanol and c h r o m a t o g r a p h e d , u s i n g a c o l u m n packed with Bondapack c 1 8 . T h e m o b i l e p h a s e c o n s i s t e d of wat e r : ethanol (52:48) containing 0. 1% N a 2 H P 0 4 and 0. 5% t e t r a b u t y l a m m o n i u m b r o m i d e , a d j u s t e d t o pH 7. 81. T h e r e c o v e r i e s f r o m p l a s m a w e r e 98. 870 f o r f l u f e n a m i c a c i d , 97.0% f o r 4 ' - h y d r o x y - d e r i v a t i v e J a n d 98.070 f o r 5-hydroxy-derivative. 6. DETERMINATION IN BODY FLUIDS AND TISSUES Many m e t h o d s a m o n g t h o s e outlined in t h i s a n a l y t i c a l p r o f i l e have been applied t o t h e detection a n d quantitative d e t e r m i n a t i o n of flufenamic a c i d in biological s a m p l e s of a n i m a l o r human origin. Such a p p l i c a t i o n s w e r e r e p o r t e d i n the p a p e r s l i s t e d below : Colorimetry 46 Spe c t rophot o m e t r y 4, 30, 40,41, 44, 60, 61 Fluorimetry 4, 8, 11, 37, 40, 45, 47, 49, 62, 63, 64, 65, 67, 69 18 Indirect A A A n a l y s i s PC 67 4, 8,40, 41, 44,45, 63, 64, 68, 6 9 TLC

FLUFENAMIC ACID

343

43, 70, 7 1 42, 72, 7 3

GC HPLC 7. AC KNOWL ED G EM EN T

T h e a u t h o r s would l i k e t o t h a n k D r . M i c h e l e L i g u o r i and M r . Vincenzo Migliaro, Ente F a r m a c o l o g i c o Italiano, N a p l e s , f o r p r o v i d i n g IR, NMR a n d UV s p e c t r a of f l u f e namic acid. 8. R E F E R E N C E S

1. M e r c k Index, 9th e d . , 1976, M e r c k a n d C o . , R a h w a y , N. J . ; p. 4028. 2. K. H. B o l t z e a n d H. K r e i s f e l d , A r z n e i m . - F o r s c h . 27, 1 3 0 0 (1977). 3. B. U n t e r h a l t , A r c h . P h a r m . 303, 4 4 5 (1970). 4. H. D. D e l l a n d R. K a m p , A r c h . P h a r m . 303, 785 (1970). 5. K. Ikeda, K. U ekam a, M. O t a g i r i a n d M. H a t a n o , J. P h a r m . Sci. 63, 1168 ( 1 9 7 4 ) . 6. J. K r a c m a r , M. A l v a r e z s o t o l o n g o , J. K r a c m a r o v a , B. M o r a v c o v a a n d H. D osl ova, P h a r m a z i e 33, 659 (1978). 29, 1 9 7. J. K r a c m a r and J. K r a c m a r o v a , C e s k . F a r m . (1980). C . A . 93, 192094 ( 1 9 8 0 ) . D o e r s i n g , W. F i s c h e r , J. F i e d l e r , 8. H. D. Del l , H. J a c o b i a n d R. K a m p , A r z n e i m . - F o r s c h . 31, 9 (1981). 9. A. C. M e h t a a n d S. G. S c h u l m a n , T a l a n t a 20, 702 (1973). 10. J. N . M i l l e r , D. L. P h i l l i p s , D. T. B u r n s a n d J. W. B r i d g e s , T a l a n t a 25, 4 6 (1978). 11. H. D. D e l l a n d B. K u x c h b a c h , F r e s e n i u s ' Z.Ana1. Chem. 262, 356 (1972). 12. J. H. Wi l ki nson a n d I. L. F i n a r , J. C h e m . S o c . , 3 2 ( 1948). 1 3 . P a r k e , D a v i s & Co. , F r . P a t e n t M1341, J u l y 2, 1962. C . A . 58, 10130a (1963). B r i s t o c M y e r s C o . , Neth.App1. 6, 604, 860, O c t . 13, 14. 1966. C . A . 66, 55513 ( 1 9 6 7 ) . 15. P. F. J uby, T. W. H u d y m a a n d M. B r o w n , J . M e d .

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M.

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16. 17. 18.

C h e m . 11, 111 ( 1968). M. K u h z r t - B r a n d s t g t t e r , A . K o f l e r a n d G. K r a m e r , S c i . P h a r m . 42, 150 ( 1 9 7 4 ) . C . A . 82, 4 7 6 8 1 ( 1 9 7 5 ) . E . M. J o n e s T P a r k e , D a v i s & C o . , B r i t . P a t e n t 953, 741, A p r . 2, 1964. C. A . 60, 1 5 6 8 8 b ( 1 9 6 4 ) . T. Minamikawa, K. Sakai, N. H a s h i t a n i , E . F u k u s h i m a and N. Y a m a g i s h i , C h e m . P h a r m . B u l 1 . 21, 1 6 3 2 (1973). R . B. Moffett and B. D. A s p e r g r e n , J. Am. C h e m . SOC. 82, 1600 (1960). A . J . A g u i a r a n d R. J. F i f e l s k i , J . P h a r m . S c i . 55, 1 3 8 7 (1 9 6 6) . A . H. Ghanem, H. E l - S a b b a g h a n d H. A b d e l - A l i m , P h a r m . Ind. 42, 854 (1980). N. A . D a a b i r S. A . K h a l i l a n d V. F. N a g g a r , Can. J. P h a r m . Sci. 11, 1 1 4 ( 1 9 7 6 ) . W. J. Dunn, J-Med.Chem. 16, 4 8 4 ( 1 9 7 3 ) . H. T e r a d a , S. M u r a o k a a n d T. F u j i t a , J . M e d . C h e m . 1 7 , 330 (1974). G. L o m b a r d i n o , I. G. O t t e r n e s s a n d E. H. W i s e m a n , A r z n e i m . - F o r s c h . 25, 1 6 2 9 ( 1 9 7 5 ) . M. K u h n e r t - B r a n d s t a t t e r , L. B o r k a a n d G. F r i e d r i c h - S a n d e r , A r c h . P h a r m . 307, 8 4 5 (1974). J. K r c , J r . , M i c r o s c o p e 25, 31 ( 1 9 7 7 ) . Z . Galdecki, M. L. G l o w k a a n d Z . G o r k i e w i c z , A c t a P o l . P h a r m . 35, 77 (1978). C.A. 89, 1 5 5 7 6 3 ( 1 9 7 8 ) . A . B u r g e r a n d R. R a m b e r g e r , M i k r o c h i m . A c t a 1, 17 (1980). H. H. F r e y a n d M. A . E l - S a y e d , A r c h . I n t . P h a r m a codyn. T h e r . 230, 300 (1977). A . A l b e r t a n d E . P. S e r j e a n t i n "The D e t e r m i n a t i o n of Ionization Constants", C h a p m a n a n d Hall, London, 1971, p. 72. H. T e r a d a a n d S. M u r a o k a , M o l . P h a r m a c o 1 . 8, 9 5 (1 9 7 2 ). U. Jahn and T. W a g n e r - J a u r e g g , A r z n e i m . - F o r s c h . 24, 4 9 4 (1974). H. M o r i y a m a , H. N a g a t a and T. T a m a k i , S u m i t o m o C h e m i c a l Co. , L t d . , J a p a n . P a t e n t 7 1 14, 656, A p r . 20, 1971. C.A. 75, 48703 (1971 ) . S. K o m u r a , K. K k a m u r a and M. T a k e n a k a , O t s u k a

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19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34.

35.

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FLUFENAMIC ACID

36.

37. 38. 39.

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

53. 54. 55.

345

C h e m i c a l D r u g s C o . , L t d . , J a p a n . P a t e n t 7 3 26, 744, A p r . 9, 1973. C.A. 79, 31680 ( 1 9 7 3 ) . G. R . Allen, J r . , and F. R . R . Church, A m e r i c a n Cyanamid Co., S. A f r i c a n P a t e n t 71 00, 512, Sep. 3, 1971. C.A. 76, 140237 ( 1 9 7 2 ) . A . J. Glazko, Ann. P h y s . Med. , Suppl. 9, 24 ( 1 9 6 7 ) . R . E. O b e r , K. Ritchie and S. F. Chang, F e d . P r o c . 24, 547 ( 1 9 6 5 ) . R. E. Bowman, K. D. Brunt, K. E. Godfrey, L. K r u s z y n s k a , A . A . Reynolds, R . I. T h r i f t , D. Waite and W. R . N. Williamson, J. Chem.Soc. P e r k i n I, 1 (1973). P. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . F o r s c h . 21, 1 6 0 5 ( 1 9 7 1 ) . H. A . R o s e n b e r g and T. R . B a t e s , P r o c . S o c . E x p . Riol. Med. 145, 93 ( 1 9 7 4 ) . C. K. Lin, C. S. Lee and .J. H. P e r r i n , J . P h a r m . Sci. 69, 95 ( 1 9 8 0 ) . L. C z e l l e s s a , R . Riva, P. Salva, F. M a r c u c c i and E. Mussini, J. C h r o m a t o g r . 192, 4 4 1 ( 1 9 8 0 ) . p. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . F o r s c h . 24, 1 2 9 8 ( 1 9 7 4 ) . H. D. Dell, J. F i e d l e r , H. J a c o b i and B. Wasche, A r z n e i m . - F o r s c h . 27, 1 3 2 2 (1977). H. D. D e l l and J. F i e d l e r , F r e s e n i u s ' Z.Ana1. Chem. 270, 278 ( 1 9 7 4 ) . D. L o r e n z and H. D. Dell, Naunyn-Schmiedeberg's A r c h . P h a r m a c o l . 277, Suppl. , R 4 4 (1973). J. A r i a s and R . Cadorniga, Boll. Chim. F a r m . 112, 804 (1973). L. Angelucci, €3. P i e t r a n g e l i , P. C e l l e t t i and S. F a villi, .J. P h a r m . Sci. 65, 455 ( 1 976). C. F. Chignell, 1 , i f e S c i . 7, 1181 (1968). C. F. Chignell, Mol. P h a r m a c o l . 5, 4 5 5 ( 1 9 6 9 ) . C. F. Chignell and D. K. S t a r k w e a t h e r , M o l . P h a r m a c o l . 7, 229 ( 1 9 7 1 ) . J. B l a n c h a r d , T . N. T o z e r , D. L. S o r b y and L. D. Tuck, Mol. P h a r m a c o l . 11, 1 3 3 ( 1 9 7 5 ) . M. Otagiri, G. E. H a r d e e and J. H. P e r r i n , Bioc h e m . P h a r m a c o l . 27, 1 4 0 1 (1978). G. Sudlow, D. J. B i r k e t t and D. N. Wade, Mol.

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62. 63. 64. 65. 66. 67. 68. 6 9. 70. 71.

72. 73.

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P h a r m a c o l . 11, 824 (1975). G. Sudlow, T J . B i r k e t t a n d D. N. W a d e , Mol. P h a r m a c o l . 1 2 , 1052 ( 1976). Y. Ka n e o, A. K ai , S. K i r y u a n d S. I g u c h i , Y a k u g a k u 86, 1 0 0 7 4 7 ( 1 9 7 7 ) . Z a s s h i 96, 1412 (1976). C.A. G. D e v a G , P. M e s n a r d a n d A . M. B r i s s o n , Ann. P h a r m . F r . 27, 239 (1969). M. I. W a l a s r a n d M. R i z k , I nd i a n J . P h a r m . 39, 8 2 (1 9 7 7 ). C . A . 87, 157253 (1977). J. B. C a r e y , J. C l i n . I n v e s t . 40, 1 0 2 8 ( 1 9 6 1 ) . Y. A . B e l t a g y , Z e n t r a l b l . P h a r m . , P h a r m a k o t h e r . Lab o r a t o r i u m s d i a g n . 116, 925 (1977). C. A , 88, 1 5 8 5 4 8 (1978). R. A . B u chanan, C. J. E a t o n , S. T. Koeff a n d A . W. K r i n k e l , C u r r . T h e r . R e s . 11, 5 3 3 ( 1 9 6 9 ) . H. D. Del l , J. F i e d l e r a n T B . W z s c h e , A r z n e i m . F o r s c h . 27, 1312 ( 1977). H. D. Del l , H. Jacobi, R. K a m p a n d J. K o l l e , A r z n e i m . - F o r s c h . 31, 2 1 ( 1981). Y. H a t t o r i , T. Arai, T. M o r i a n d E. F u j i h i r a , C h e m . P h a r m . Bull. 18, 1 0 6 3 (19 7 0 ) . L. R e io , J. C h r o m a t o g r . 68, 1 8 3 ( 1 9 7 2 ) . W. S c h m o l l a c k a n d U. W e n z e l , P h a r m a z i e 29, 5 8 3 (1 9 7 4 ). B. D e m e t r i o u a n d B. G. O s b o r n e , J. C h r o m a t o g r . 90, 4 0 5 (1 9 7 4) . H. E. G e i s s l e r , E. M u t s c h l e r a n d A . S c h u m a c h e r , J. C h r o m a t o g r . 146, 1 6 9 ( 1978). H. R o s e b o o m a n d A . Hulshoff, J. C h r o m a t o g r . 173, 6 5 (1 9 7 9 ) . J. P. Hunt, P. E. Haywood a n d M. S. M o s s , P r o c . Int. S y mp . E q u i n e Med. C o n t r o l , 3 r d , 1979, 9. C. A . 94, 97262 (1981). J. D u s c i a n d L. P. H a c k e t t , J. C h r o m a t o g r . 1 7 2 , 5 1 6 (1 9 7 9) . 0. Kubo, K. N i s h i d e a n d N. K i r i y a m a , J. C h r o m a t o g r . 174, 254 (1979).

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1981.

HEXESTROL Hassan X Aboul-Enein, Essam A. Lo@, and Mohumed E . Mohumed

348 348 348 348 349 349 349 349 349 350 350 350 358 361 361 362 362 362 363 364 370 370 372

1. Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight I .4 Elemental Composition 1.5 Appearance, Color, Odor 2. Physical Properties 2.1 Crystal Properties 2.2 Melting Point 2.3 Solubility 2.4 Identification 2.5 Spectral Properties 3. Synthesis 4. Stability and Decomposition Products 5 . Metabolism 6. Methods of Analysis 6.1 Titrimetry 6.2 Colorimetry 6.3 Ultraviolet Spectrophotometry (Uv) 6.4 Chromatographic Analysis 6.5 Mass Fragmentography 6.6 Biological Assays References

Analytical Profiles of Drug Substances Volume I I

347

Copyright 0 1982 by The American Pharmaceutical hsociation

ISBN 0-12-Mo811-9

HASSAN Y. ABOUL-ENEIN ET AL.

348

ANALYTICAL PROFILE-HEXESTROL

1. Descr i p t ton 1.1 Nomenclature 1.11 - Chemical N a m e s (?)

3 , 4 - Di(p-hydroxy phenyl) n-hexane. 4 , 4 ' - (1, 2 d i e t h y l - 1, 2 - e t h a n e d i y l ) bis-phenol. 4 , 4 ' - (1, 2 d i e t h y l e t h y l e n e ) d i p h e n o l . 4 , 4 ' - dihydroxy - y,b-diphenyl hexane. 4 , 4 ' - dihydroxy - a ,B-diethyldiphenylethane. p, p l - dihydroxy - d i p h e n y l hexane. meso - 3 , 4 - b i s (p-hydroxyphenyl) -nhexane

.

1 . 1 2 - Generic Name H e x e s t r o l , H e x o e s t r o l , Dihydrodiethyls t i l b e s t r o l , Hexanoestrol, Cycloestrol , Hormoestrol, S y n e s t r o l .

1.13 - Trade Name S yn t r o g h e , Fo 11i p l e x , S yn t hovo

1.2

Formula

1 . 2 1 - Emprical

'18 H22 '2 1.22

- Structural C2R5

1.3

Molecular Weight 270.4

.

349

HEXESTROL

1.4

Elemental Composition C , 79.96%; H, 8.2%; 0,11.84%

1.5

Appearance, Color, odor w h i t e , o d o r l e s s , c o l o r l e s s c r y s t a l s or c r y s t a l l i n e powder (1).

2.

Physical properties

2.1

Crystal properties The f o l l o w i n g t a b l e shows t h e c r y s t a l form of h e x e s t r o l d e r i v a t i v e s and t h e i r m e l t i n g p o i n t s . These d e r i v a t i v e s c a n be used f o r i d e n t i f i c a t i o n purposes too ( 2 ) .

Derivative

Crystalization solvent

0

Crystal form

M.P.,

plates crystals crystals crystals crystals

146 127-128 106-107 96-97 150-153

C

Meso Form Di-Me e t h e r Dipropionyl Dibutyryl Dicaproyl Disuccinyl

Me2CO-MeOH Pet. ether Pet. ether

Pet. e t h e r CHC13-pet. e t h e r

DL(+) Form Di-Me

2.2

ether

C&-pet.

ether

56

Melting p o i n t The f o l l o w i n g are t h e m e l t i n g p o i n t s f o r h e x e s t r o l i n i t s meso, DL-forms and t h e i r a n t i p o d e s .

Form M.P.,

0

Ref

C

Meso -

DL (+)Form

185-188 184-185 186

3 4 5

128

2

350

HASSAN Y. ABOUL-ENEIN ET AL. L(-) Form D(+) Form

2.3

80 80

2 2

Solubility Freely soluble i n ether, soluble i n acetone, a l c o h o l , methanol, s o l u b l e i n v e g e t a b l e o i l s upon warmming a l s o s o l u b l e i n d i l u t e s o l u t i o n of a l k a l i h y d r o x i d e . S l i g h t l y s o l u b l e i n c h l o r o f o r m , benzene; i n s o l u b l e i n water (1, 3 ) .

2.4

Identification The f o l l o w i n g i d e n t i f i c a t i o n tests a r e d e s c r i b e d i n t h e P h a r m a c e u t i c a l Codex 1979 ( 5 ) .

1) To a b o u t 25Omg add l m l of a c e t i c a n h y d r i d e and 2 m l of d e h y d r a t e d p y r i d i n e , b o i l u n d e r a r e f l u x c o n d e n s e r f o r 1 5 m i n u t e s , c o o l , add 5Oml of water and s h a k e t h o r o u g h l y u n t i l a p r e c i p i t a t e is produced which, a f t e r washing w i t h water and d r y i n g , m e l t s a t a b o u t 138OC. 2) D i s s o l v e a b o u t lOmg i n 5 m l of H2SO4; t h e s o l u t i o n i s c o l o r l e s s ( d i s t i n c t i o n from s t i l b o e s t r o l , which g i v e s a g o l d e n - y e l l o w color). 2.5

Spectral properties

2.51

Ultraviolet spectra H e x e s t r o l i n e t h a n o l shows maxima a t 230nm ( E l % , l c m , 775) and 280 nm ( E l % , lcm, 1 4 0 ) ; 0.11NaOH h e x e s t r o l g i v e s maxima a t 242 nm ( E l % , l c m , 965) and 295 nm (El%, l c m , 1 7 5 ) . A s shown i n f i g u r e ( 1 ) and i n agreement t o t h e f i g u r e s p u b l i s h e d by C l a r k e ( 3 ) .

2.52

Infrared Spectra The i n f r a r e d spectrum of h e x e s t r o l i n K B r d i s c i s g i v e n i n f i g u r e ( 2 ) . Major band a s s i g n m e n t s are a s follows: -1 Assignment Frequency Cm 3400 P h e n o l i c OH 1610, 1600 Aromatic r i n g C=C s t r e t c h

35 1

HEXESTROL

L 200 nm

F i g . 1.

300 nm

250 nm

350 nm

The u l t r a v i o l e t a b s o r p t i o n spectrum of H e x e s t r o l

i n ethanol. I n s t r u m e n t : Pye Unicam SP8-100

Wavenumber

Figure 2.

I R s p e c t r u m of H e x e s t r o l i n K B r .

Instrument:

P e r k i n Elmer 567

HASSAN Y. ABOUL-ENEIN ET AL.

352

Other f i n g e r p r i n t bands c h a r a c t e r i s t i c to-l h e x e s t r o l a r e 1530, 1450, 1300 and 1110 cm 2.53 a)

.

Nuclear Mametic Resonance Spectrum PMR A t y p i c a l PMR spectrum of h e x e s t r o l i s shown

i n Figure (3). The sample was d i s s o l v e d i n d e u t r a t e d CDC13 and a drop of d e u t r a t e d d i m e t h y l s u l f o x i d e (DMSO-db) u s i n g TMS a s t h e i n t e r n a l s t a n d a r d . The spectrum was determined on a V a r i a n T60-A s p e c t r o m e t e r . The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have been e l i c i t e d from F i g u r e ( 3 ) . Chemical S h i f t (6)

T r i p l e t a t 0.50

CH CH -3 2

Multiplet centered a t 1.3

C H e 2

M u l t i p l e t c e n t e r e d a t 1.90

b)

Assignment

-CH-CH-

Doublet of d o u b l e t c e n t e r e d a t 6.86

eight aromatic protons character i s t i c f o r para substitution of t h e r i n g .

Broad s i n g l e t exchangable w i t h D20 a t 7.67

p h e n o l i c OH group.

13C-NMR

*

The I3C-NMR of h e x e s t r o l has been d e t e r m i n e d , t h e off-resonance spectrum ( F i g . 4 ) shows seven s i n g l e t s . The complete spectrum i s shown i n F i g . 5. The spectrum was determined on a Varian FT-80A i n DMSO-db a s s o l v e n t , t u b e d i a m e t e r 10 mm, s p e c t r a l width 5000 Hz

*H.Y. Aboul-Enein, unpublished d a t a

I

I

.

1

ao

.

.

F i g u r e 3.

.

.

1

ZO

.

I

I

.

.

.

1

6.0

.

.

.

.

1

5.0

.

.

. . ~ PPM( b) 4.0

.

.

. .

.

3.0

1

~

.

.

.

. ’ 2.0

~

”

. ” 1.0

PMR s p e c t r u m of H e x e s t r o l i n C D C l -DMSO-d w i t h IT% a s i n t e r n a l standard. 3 6

Instrument:

V a r i a n T60-A

”

~

354

355

356

HASSAN Y. ABOUL-ENEIN ET AL. a c q u i s a t i o n t i m e : 1.638 sec.; p u l s e w i d t h : 4 Usec; number of d a t a p r i n t : 16384. S p e c t r a l a s s i g n m e n t s a r e l i s t e d below:

(

Chemical S h i f t i n ppm r e l a t i v e t o TMS) 12.06

CH3

26.97

CH2

52.77

CH-

134. 4 2 115.05 128.82 155.39 2.54

Assignments

c 1- c.1 c 2-c 6 02- c 6 c3-c 5 c--c 3 5 c4-c*4

Mass Spectrum

The mass spectrum of h e x e s t r o l , o b t a i n e d by chemical i o n i z a t i o n w i t h i s o b u t a n e g a s , i s shown i n P i g . 6. The spectrum w a s determined by d i r e c t i n l e t t o Ribermag 10-10R m a s s s p e c t r o m e t e r and e x h i b i t s c o m p a r a t i v e l y l i t t l e fragmentation. The f o l l o w i n g t a b l e g i v e s t h e most prominent i o n s and t h e i r r e l a t i v e i n t e n s i t i e s .

Mass ( d e )

27 0

Relative Intensity % 4.6 (M+)

219

10.5

178

11.8

1 77

93

1 36

9.9

. I

80 90

14011012Q 130 W 1 5 0 1601?01801902002102PO2303+0250260270

F i g u r e 6.

Mass Spectrum of H e x e s t r o l ( C I - i s o b u t a n e ) by d i r e c t i n l e t i n s e r t i o n .

determined

HASSAN Y. ABOUL-ENEIN ET AL.

358

100 ( b a s e peak)

135 134

36.9

107

42.6

+'

+.

1

0

-@

CH2CH3

I

CH-CH m/e 177

mfe 178 +*

CH2CH3

O D

CH2CH3

H@ @ H!

LH

m f e 134

m f e 135 3.

CH CH r - C Hl 2

Synthesis S e v e r a l methods have been p u b l i s h e d and p a t e n t e d f o r t h e s y n t h e s i s of h e x e s t r o l . I n 1938 Campbell et-a1 - (6) i s o l a t e d h e x e s t r o l i n p o o r y i e l d , from t h e p r o d u c t s of d e m e t h y l a t i o n of a n e t h o l e . Some of t h e s y n t h e t i c a p p r o a c h e s f o r h e x e s t r o l a r e summerized a s f o l l o w s : -

1) C a t a l y t i c h y d r o g e n a t i o n of p s e u d o d i e t h y l s t i l b e s t r o l g i v e s h e x e s t r o l a l o n g w i t h some d i e t h y l s t i l b e s t r o l (7).

=:

EtEt

H

O

G

@OH

I

catalytic hydrogenat i o n

HO&-

,CH

I

Et

@H+HO

0

Et

I c = c I

Et

+*

359

HEXESTROL Hydrogenation of t h e d i m e t h y l e t h e r s of d i e t h y l s t i l b e s t r o l and pseudodiethylstilbestrol w i t h subsequent d e m e t h y l a t i o n a f f o r d s t h e meso-isomer of h e x e s t r o l which i s more p o t e n t t h a n t h e DL-isomer ( 7 ) . H CH CH

HO

meso-f orm 2) Hydrogenation of 4 , 4 ' dihydroxy y-6 d i p h e n y l y-6 hexadiene g i v e s h e x e s t r o l i n q u a n t i t a t i v e y i e l d ( 7 ) .

CH CH

HOO'L!!

0

Fd Icharcoa 1

>- .

OH

Hexestrol

3) H e x e s t r o l may be prepared by t h e a c t i o n of e t h y l magnesium bromide on a n i s a l d a z i n e w i t h subsequent d e m e t h y l a t i o n (8).

Hexes t r o l The above methods f o r t h e s y n t h e s i s of h e x e s t r o l i n v o l v e t h e u s e of i n t e r m e d i a t e s which a r e d i f f i c u l t t o prepare.

360

HASSAN Y. ABOULENEIN ET AL. 4) B e r n s t e i n and Wallis d e s c r i b e d ( 4 ) a n a l t e r n a t e i n e x p e n s i v e method f o r t h e s y n t h e s i s o f h e x e s t r o l s t a r t i n g from p-hydroxypropiophenone a s shown i n scheme ( 1 )

I I1

I1

-@

-b ,CH30 N a

YHCH

alcohol

OH2

I11 I11

F

HBr C

H

3

@'

0

CH-CH

Br 2 5

IV

Scheme 1

VI

5) Kharasch and Kleiman(9) p r e p a r e d h e x e s t r o l d i m e t h y l e t h e r from a n e t h o l e hydrobromide and G r i g n a r d r e a g e n t i n t h e p r e s e n c e of a h a l i d e of c o b a l t , n i c k e l o r i r o n , t h e f r e e r a d i c l e g e n e r a t e d from t h i s r e a c t i o n dimerizes t o give hexestrol dimethylether i n y i e l d s r a n g i n g from 14-41%. The h i g h e r m e l t i n g p o i n t f o r t h e meso-form a l l o w s i t s s e p a r a t i o n from t h e DLby-product formed i n t h i s r e a c t i o n .

r

1

HEXESTROL

4.

361

S t a b i l i t y and Decomposition p r o d u c t s H e x e s t r o l is a r e l a t i v e l y s t a b l e compound a t room t e m p e r a t u r e ; however i t i s recommended t o b e k e p t i n a w e l l c l o s e d c o n t a i n e r p r o t e c t e d from l i g h t .

5.

Yetabolism I l e x e s t r o l i s m e t a b o l i z e d i n a s i m i l a r way a s d i e t h y l s t i l b e s t r o l , i t i s excreted c h i e f l y as a glucuronide conjugate ( 3 ) . This glucuronide i s mostly excreted i n t o t h e b i l e which i s s u b j e c t e d t o h y d r o l y s i s by i n t e n t i n a l g l u c u r o n i d a s e enzyme d u r i n g i t s p a s s a g e i n t o t h e g u t . T h i s a l l o w s t h e d r u g be r eab so r b ed , r e c o n j uga t e d and r e - e x e r e t e d ( h e p a t i c c i r c u l a t i o n ) ( 1 0 ) . O t h e r p o s s i b l e m e t a b o l i t e i n t e r m e d i a t e s which This should b e i n v e s t i g a t e d a r e shown in scheme ( 2 ) . i s i n a n a l o g y t o t h e p o s s i b l e m e t a b o l i t e s of d i e t h y l s t i l b e s t r o l which h a s r e c e n t l y a t t r a c t e d t h e a t t e n t i o n by b e i n g l i n k e d t o t h e o c c u r a n c e o f v a g i n a l a d e n o c a r c i noma i n a d o l e s c e n t d a u g h t e r s whose m o t h e r s had r e c e i v e d d i e t h y l s t i l b e s t e r a l d u r i n g p r e g n a n c e (11, 1 2 , 13).

HO HO *

H

o

e

o OH

Me0

Me0

HO

Me0

Scheme 2.

Expected M e t a b o l i t e s of H e x e s t r o l

H

HASSAN Y. ABOUL-ENEIN ET AL.

6.

Methods of A n a l y s i s

6.1

Titrimetry

- Aqueous (Bromometry) The f o l l o w i n g method h a s been d e s c r i b e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of h e x e s t r o l d i a c e t a t e (14) : To 70 mg of sample add 1 0 m l of 0.5N m e t h a n o l i c KOH, and h e a t under r e f l u x f o r 30 m i n u t e s on a water-bath. Cool, add 5Oml of a c e t i c a n h y d r i d e , shake u n t i l t h e h y d r o l y s a t e i s d i s s o l v e d , t h e n add 2 m l of 30% K B r s o l u t i o n , 2 ml of conc. H2SO4 and 20 m l of 0.1N-KBr03, and set a s i d e i n t h e d a r k f o r 1 0 min. Add lgm of K I and 100 m l of H 2 0 , and t i t r a t e w i t h O.lN-Na2S203 u s i n g +1% over t h e r a n g e s t a r c h a s i n d i c a t o r . The e r r o r i s 7 0 t o 90 mg of h e x e s t r o l d i a c e t a t e . 6.2 Colorimetry H e x e s t r o l has been determined i n o i l y s o l u t i o n and t a b l e t form a s f o l l o w s ( 1 5 ) : For t a b l e t : e x t r a c t a sample c o n t a i n i n g 2 t o 3 mg of h e x e s t r o l w i t h methanol, d i l u t e t h e e x t r a c t w i t h water t o 100 m l , and f i l t e r . To a 20 m l a l i q u o t add b o r a t e b u f f e r s o l u t i o n (pH 1 1 ) . For o i l y i n j e c t i o n s : Mix a sample c o n t a i n i n g 0.4 t o 0.6 mg of h e x e s t r o l w i t h l i g h t petroleum (5ml) , shake t h e s o l u t i o n w i t h methanol (6ml) , add b u f f e r s o l u t i o n (13ml), shake, and c o l l e c t t h e aq. methanol phase i n a lOOml f l a s h . Twice r e p e a t t h e e x t r a c t i o n w i t h methanol (6ml) and b u f f e r s o l u t i o n (13ml) and t o t h e combined aq. e x t r a c t s add methanol (2ml). For d e t e r m i n a t i o n of h e x e s t r o l : h e a t t h e prepared s o i u t i o n on a water b a t h f o r 30 min., c o o l i t t o room temperature, add d i a z o t i z e d s u l p h a n i l i c a c i d s o l u t i o n (12ml) and mix. A f t e r 40 minutes d i l u t e w i t h b u f f e r s o l u t i o n t o 100 m l , and measure t h e e x t i n c t i o n a t 495 nm a g a i n s t water. Beer's law i s obeyed f o r 0.5 t o 4 mg of h e x e s t r o l p e r 1 O O m l . R e s u l t s a g r e e to. w i t h i n +lo% w i t h t h e o r e t i c a l values.

HEXESTROL

363

B e l i k o v , d e s c r i b e d a s i m i l a r p r o c e d u r e (16) which depends on d i a z o d i z a t i o n of h e x e s t r o l w i t h d i a z o s u l p h a n i l i c a c i d . However, t h e azo-dye formed i s measured i n a l k a l i n e medium a t 420 nm. Another c o l o r i m e t r i c method f o r d e t e r m i n a t i o n of hexestrol i n feeds i s reported a s follows (17): The ground f e e d i n g s t u f f (40gm) mixed w i t h lOgm of sand i s s e t a s i d e w i t h c h l o r o f o r m o v e r n i g h t , t h e n e x t r a c t e d w i t h CHC13 f o r 6 h o u r s . The e x t r a c t i s made up t o 2 O O m l of CHC13. The r e s i d u e from e v a p o r a t i o n of t h e f i n a l CHC13 e x t r a c t i s d i s s o l v e d i n t r i e t h y l a m i n e t e t r a h y d r o f u r a n - w a t e r (1:5 : 1 4 ) , t h e s o l u t i o n is r e t a i n e d i n an o l e a t e d c e l l u l o s e column f o r 1 h o u r , t h e n t h e impurities a r e eluted with triethylamine - tetrahydrof u r a n - water m i x t u r e . The column i s a c i d i f i e d w i t h N-H2SO4 and e x t r a c t e d w i t h e t h y l e t h e r , t h e e x t r a c t i s evaporated, the r e s i d u e i s dissolved i n ethanol, and t o t h i s s o l u t i o n a r e added water, dil.HC1, a molybdotungstophosphate r e a g e n t and t h e n Na2C03 s o l u t i o n . The e x t i n c t i o n of t h e c e n t r i f u g e d s o l u t i o n i s measured a t 750 run a g a i n s t a r e a g e n t b l a n k and compared w i t h t h a t of s t a n d a r d s o l u t i o n of h e x e s t r o l t r e a t e d s i m i l a r l y .

6.3

U l t r a v o i l e t S p e c t r o p h o t o m e t r y (U.V.)

H e x e s t r o l w a s q u a n t i t a t i v e l y determined i n tablet s p e c t r o p h o t o m e t r i c a l l y ( 1 8 ) . The d r u g was e x t r a c t e d w i t h CHC13 from a n a c i d i f i e d powdered sample ( c o n t a i n i n g a b o u t 5mg of h e x e s t r o l ) . The CHC13 e x t r a c t w a s conc e n t r a t e d and 2 , 2 , 4 t r i m e t h y l p e n t a n e w a s added and h e x e s t r o l w a s e x t r a c t e d w i t h 0.1N NaOH. The combined a l k a l i n e s o l u t i o n w a s a c i d i f i e d and r e - e x t r a c t e d w i t h CHC13. The CHC13 s o l u t i o n was washed w i t h water and d r i e d o v e r Na2S04 and e v a p o r a t e d t o d r y n e s s . The r e s i d u e w a s d i s s o l v e d i n e t h a n o l and measured a t 280nm. The p e r c e n t a g e r e c o v e r y ranged from 94.4-98.9%. Kovalenko r e p o r t e d a q u a n t i t a t i v e method f o r t h e a s s a y of h e x e s t r o l ( 1 9 ) . About 25mg of h e x e s t r o l w a s weighed and d i s s o l v e d i n 25-3Om1 of 0.1N NaOH i n a 5Oml v o l u m e t r i c f l a s k . The volume w a s a d j u s t e d t o 5Oml w i t h 0.1N NaOH s o l u t i o n . Then p i p e t t e 5ml of t h i s s o l u t i o n i n t o a second 5Oml v o l u m e t r i c f l a s k and t h e volume a d j u s t e d

HASSAN Y. ABOUL-ENEIN ET AL.

364

t o 5Oml w i t h 0.1N NaOH s o l u t i o n . The same s e r i a l d i l u t i o n was r e p e a t e d a n d t h e c o n c e n t r a t i o n of h e x e s t r o l i n t h e l a s t s o l u t i o n w a s d e t e r m i n e d by m e a s u r i n g t h e a b s o r p t i o n a t 241 nm u s i n g 0.1N NaOH s o h t i o n f o r t h e b l a n k . S i n c e t h e s e s o l u t i o n s f o l l o w t h e Lambert-Beer law 1.8 pg m l , t h e d e t e r m i n a t i o n c a n b e made from a s t a n d a r d c u r v e . 6.4

Chromatographic A n a l y s i s 6 . 4 1 P a p e r Chromatography Tompsett h a s d e s c r i b e d a method f o r d e t e c t i o n of h e x e s t r o l and o t h e r s t i l b e s t r o l d e r i v a t i v e s a l o n g w i t h t h e p - h y d r o x y m e t a b o l i t e s of p h e n o b a r b i t o n e and p h e n y t o i n i n u r i n e u s i n g t h e 2-dimensional paper chromatography ( 2 0 , 2 1 ) . The two s y s t e m s u s e d were i s o p r o p a n o l : NH3 (0.99) :H20 (8 :1:1) and C6H6 :E t O A c :H 2 0 ( 2 :1: 1). The d e t e c t i n g a g e n t s u s e d were P a u l y ' s r e a g e n t (red-brown) , d i a z o t i z e d p - n i t r o - a n i l i n e (brown) , d i a z o t i z e d d i e t h y l a m i n o e t h y l p-aminophenyl s u l p h o n e (brown) and 1 - n i t r o s o - 2 - n a p h t h o l n i t r i c a c i d m i x t u r e (+ve). The s e n s i t i v i t y r a n g e f o r t h i s method i s 5-80ug. 6.42 Column Chromatography Column chromatography h a s b e e n a p p l i e d t o p u r i f y the c a t t l e feed e x t r a c t s containing h e x e s t r o l and o t h e r s t i l b e s t r o l s t o remove i n t e r f e r i n g substances b e f o r e i t s determinat i o n . An example of t h e columns u s e d f o r t h e p u r i f i c a t i o n of t h e e x t r a c t s i s A 1 0 column 2 3 (22). Verbeke ( 2 3 ) used columns c o n t a i n i n g XAD-2, C e l i t e , o r n e u t r a l A1203 (Brockman A c t i v i t y I ) f o r p u r i f i c a t i o n o f t i s s u e e x t r a c t s and u r i n e u s i n g d i s t i l l e d water; 1 5 m l water-washed e t h e r f o l l o w e d b y , lhl CgHg t h e n benzenei s o o c t a n e ( 1 : l ) ; and b e n z e n e : i s o o c t a n e (1:l) a s e l u e n t s r e s p e c t i v e l y f o r d e t e c t i o n of h e x e s t r o l and o t h e r a n a b o l i c s .

365

HEXESTROL 6 . 4 3 Thin Layer Chromatography S e v e r a l r e p o r t s have been published on t h e d e t e c t i o n , q u a n t i t a t i v e d e t e r m i n a t i o n of h e x e s t r o l and o t h e r c h e m i c a l l y r e l a t e d d r u g s f o r example d i e t h y l s t i b e s t r o l i n f e e d s (meat, milk) and i n b i o l o g i c a l f l u i d s . Table (1) summerizes t h e s o l v e n t systems a n d t h e d e t e c t i n g a g e n t s used i n t h e cited references.

6.44 Gas-Liquid Chromatography Various g a s - l i q u i d chromatographic methods have been developed f o r d e t e c t i o n of h e x e s t r o l i n meat and o t h e r a g r i c u l t u r a l p r o d u c t s . Gain and S c h o l l ( 3 1 ) d e s c r i b e d a method f o r d e t e r m i n a t i o n of h e x e s t r o l i n m o l a s s e s - based l i q u i d feed supplements, a f t e r t h e p r e p a r a t i o n of t h e b i s ( t r i m e t h y l s i l y l ) acetamide d e r i v a t i v e . However t h e method showed i n t e r f e r i n g peaks o r low recovery due t o emulsion f o r m a t i o n . Most of t h e g a s chromatographic d e t e r m i n a t i o n r e q u i r e s d e r i v a t i z a t i o n of h e x e s t r o l b e f o r e i n j e c t i n g i n t o t h e g a s chromatograph. Table ( 2 ) summerizes t h e d a t a o b t a i n e d from t h e l i t r a t u r e s t i l l 1980.

T a b l e 1.

S o l v e n t system

a)

Stationary phase

Detect i n p agent

m m

R-emarks

2 d i m e n s i o n a l TLC

5%H2SO4-

s e n s i t i v i t y 0.5-

CHC13:EtOH:C H 6 6 (36:1:4)

induced f l u o r e

lOppb

Ref.

23

scence a t

n-C H :Et20:CH2C12 6 14 (4:3: 2)

W

(contd.)

366m v a n i l li n

petroleum e t h e r

Silica gel

(40-65OC) :

( a c t i v a t e d a t 120 OC reagent)

a p p l i e d t o animal

27

feeding s t u f f

2 d i m e n s i o n a l TLC using: a)

CHC13:EtOH(9:1)

b)

C 6H :E t OAc ( 3 :1 )

28

Silica gel H

t.1.c CH2 C12:Me2C0(4 :1)

K i e s e l g e l or

ultraviolet a t

s e n s i t i v i t y range

Kieselgel F

254m

0 . 2 . 2 up,

254

( t h e h i g h performance t l c r a n g e 10-200ng

29

T a b l e 1.

S o l v e n t system

Toleune: EtOAc ( 1 9 :1 )

Stationary phase

A l u m i n ium ox i d e

Detecting agent

Ultraviolet a t 254 nm

C6H6 :E t O A c (20: 1)

CH2C12:Me2C0(4: 1)

Remarks

S e n s i t i v i t y 5,lOppb

Ref.

24

( i n f r e s h l i v e r and kidney) Silica gel

U 1 tr av i o l e t a t 254 nm

recommended € o r r o u t i n e test of e s t r o g e n s i n food

25

Silica gel G

ultraviolet

s e n s i t i v i t y of t h e

26

254m. por 20'

test i s 0 . 5 y g

CHC13:EtOAc(4:1)

2 d i m e n s i o n a l TLC w i t h n C6H14:Et20:CH2C12

(4:3:2) i n b o t h d i r e c t i o n or EtOAc:C6H6(l:3) a f t e r s o l v e n t system ( a ) n-C H

6 14 ( 4 :3 :2)

:Et20:CH2C12

T a b l e 1.

S o l v e n t system

Stationary phase

(contd.)

Detecting agent

Remarks

Ref.

h.p. t.1.c.

CH C1 :Ple2C0(4:1) 2

2

Kieselgel F

W

91 W

254

d e n s itome-

(t.1.c.

range

trically at

200-2000ng)

.

287

(h.p.t.1.c

range

10-2 Ong ) C6H14:Me2C0(3 :2 ) o r C6H6:Me2C0(3:1)

Po 1yamid e

t h e i n f l u e n c e s of chemical s t r u c t u r e and m i g r a t i o n r a t e are discussed.

30

T a b l e 2.

Carrier G a s

Column

Detector

Remarks

d er i v a t i z e d by hep t a f l u o r o b u t y r i c a n hyd r i d e

3% OV-1 on Chromosorb

Ar-CH

WHP (80 t o 100 mesh) a t

(19:l)

capture

He

electrolytic conductor

s e n s i t i v i t y €or electron c a p t u r e (40-400 p i c o g ) ; and f o r e l e c t r o l y t i c c o n d u c t o r (1-5 ng)

electron capture

d e r i v a t i z e d by heptafluorobutyric anhydride; s e n s i t i v i t y range 6 t o 262 ng/ml

flame ionizat ion d e t e c to r

i n j e c t e d as a dipropionate derivative; sensitivity 0.05 mg/ml

Electroncapture

d er i v a t i z ed by trifluoroacetic anhydr iod e .

4

electron

Ref.

32

var ious temperature

2% OV-17, 3%0V-1,3%0V-1 5% of n e o p e n t y l g l y c o l s e b a c a t e o r 2% Carbowax 20M on s i l a n i s e d Chromosorb W (80 t o 100 m e s t ) a t var i o u s temperatures

N2

32

33

.

3% QF-1 o n 100 t o 200 mesh Gas. Chrom Q a t 170'. Ap i e z o n L-Ep i k o t e 1001(5 :1) on s i l a n i s e d Chromosorb G a t 160Oc.

N2

34

3 70

HASSAN Y. ABOUL-ENEIN ET AL. 6.45 High Performance L i q u i d Chromatography H e x e s t r o l has been determined among o t h e r a n a b o l i c s y n t h e t i c and n a t u r a l hormones i n meat by HPLC. The columns used were Rp-2 (10 v m ) , Rp-18 ( 5 pm) o r Zorbax CN ( 5 pm) by g r a d i e n t e l u t i o n w i t h a c e t o n i t r i l e : H 2 0 (1:9) p l u s 25%, i n c r e a s i n g r e c t i l i n e a r l y i n 5 m i n u t e t o 45%, of a c e t o n i t r i l e : H20 ( 9 : l ) . The f l o w r a t e w a s 2 m l min.-l, and 100 mg e a c h of L i C l and LiC104 were added t o each 1 0 h l of e l u e n t . The e l u a t e w a s examined by voltammetry w i t h v i t r e o u s carbon e l e c t r o d e s and on silver-AgC1 (3M-KC1) r e f e r e n c e e l e c t r o d e ; t h e p o t e n t i a l sweep r a t e was 5 m ~ s - 1 . Other columns used f o r t h e d e t e r m i n a t i o n of h e x e s t r o l and o t h e r r e l a t e d d r u g s i s L i c h r o s o r b RP-8 w i t h m i x t u r e s (35:13, t o 25:23) of methanol and water ( c o n t a i n i n g i n a l l i n s t a n c e s 2 p a r t s of a c e t o n i t r i l e ) as a mobile phase (0.8 t o 1.6 m l min-l). The v o l t a m m e t r i c c u r v e of t h e e l u a t e was recorded by t h e u s e of a v i t r e o u s - c a r b o n e l e c t r o d e , a p l a t i n u m c o u n t e r - e l e c t r o d e and a silver-AgC1 r e f e r e n c e e l e c t r o d e a t pH 3 , t h e peak p o t e n t i a l ( V ) f o r h e x e s t r o l w a s +.9. Amounts r a n g e from 1-4 ng g-l of h e x e s t r o l i n meat could be determined by HPLC

.

6.5

Mass Pragmentography H e x e s t r o l among o t h e r s t i l b e s t r o l w a s q u a n t i t a t i v e l y determined by combined GC/MS ( 3 8 ) . The s e n s i t i v i t y r a n g e i s 40 p a r t p e r 1 09 , t h e method

is s u c c e s s f u l l y used f o r d e t e c t i o n of e s t r o g e n s and i n meat p r o d u c t s . I t i n v o l v e s e x t r a c t i o n of l i v e r , kidney and muscle t i s s u e i n a c e t o n i t i l e : water ( 9 : l ) . The drug w a s c o n v e r t e d t o i t s t r i m e t h y l s i l y l d e r i v a t i v e f o r a n a l y s i s on a column packed w i t h 2%0V-17 on Chromosorb G and coupled t h r o u g h a Watson-Biemann H e s e p a r a t o r t o t h e mass s p e c t r o m e t e r . 6.6

B i o l o g i c a l Assays Heinert, i d e n t i f i e d hexestrol i n milk using the mouse u t e r i n e weight b i o a s s a y method ( 3 9 ) . Rennet

HEXESTROL

371

c o a g u l a t i o n of t h e m i l k p e r m i t t e d d e t e c t i o n of 0.001 ppm of h e x e s t r o l which could a l s o be d e t e c t e d e ta1 (40) i n c h e e s e a f t e r c o a g u l a t i o n of m i l k . L i e m d e s c r i b e d a b i o l o g i c a l a s s a y of e s t r o g e n i c s u b s t a n c e s i n cosmetic i n c l u d i n g h e x e s t r o l , based on a p p l i c a t i o n of t h e cosmetic p r o d u c t s t o t h e shaven s k i n of c a s t r a t e d female mice; v a g i n a l smears were t a k e n subsequently f o r a n a l y s i s .

Acknowledgements

The a u t h o r s wish t o t h a n k M r . Khalid N.K. Lodhi f o r h i s technical a s s i s t a n c e i n determining t h e NMR s p e c t r a of h e x e s t r o l and M r . Uday C. Sharma f o r t y p i n g t h e manuscript.

HASSAN Y. ABOUL-ENEIN ET AL.

372

References

1.

a)

R e m i n g t o n ' s P h a r m a c e u t i c a l S c i e n c e s , #ack P u b l i s h i n g Co., E a s t o n , P a . , p. 920 ( 1 9 7 5 ) .

b)

Merck I n d e x , N i n t h e d i t i o n , Merck & Co. , I n c . , Rahaway, N . J . , U.S.A., p . 615, 4571 ( 1 9 7 6 ) .

2.

D i c t i o n a r y of o r g a n i c compounds Vol. 2 , E y r e S. S p o t t i s woods - (London) P u b l i s h e r s L t d . , E . & F. M. Spon L t d . , New York, p. 1036 ( 1 9 6 5 ) .

3.

E.G.C. C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs", The P h a r m a c e u t i c a l P r e s s , London, 1 9 6 9 , p. 363.

4.

S. B e r n s t e i n and E.S.

Wallis, J . Amer. Chem. S O C . ,

62, 2871, ( 1 9 4 0 ) . 5.

6.

The P h a r m a c e u t i c a l Codex, E l e v e n t h E d i t i o n , t h e P h a r m a c e u t i c a l Press, London, p . 4 1 1 ( 1 9 7 9 ) . N.R.

Campbell, E . C .

Dodds and W . Lawson, N a t u r e ,

142, 1 1 2 1 ( 1 9 3 8 ) .

7.

E . C . Dodds, R . Robinson, e t a l . ( a ) N a t u r e , 141, 247 (1938), (b) P r o c . Roy. SOC. (London), B 7 , 1 4 0 (1939).

8.

N . R . Campbell, E.C. Dodds and W. Lawson, P r o c . Roy. SOC. (London), B 8 , 253 ( 1 9 4 0 ) .

9.

M.S.

K h a r a s c h and M. Kleiman, J . Amer. Chem. SOC. (1943).

65, 491,

10.

B.N. La Du, H.G. Mandel and E . L . Way, "Fundamentals of Drug Metabolism and Drug D i s p o s i t i o n " , The W i l l i a m s & W i l k i n s Co., B a l t i m o r e , MD., 1 9 7 2 , p.263.

11. A.L. H e r b s t , H. U l f e l d e r and D . C . P o s k a n z e r , N e w Engl. J . Med.,

284,

878 ( 1 9 7 1 ) .

12.

A.L. H e r b s t , R . J . Kurman and R . E . GynecFl. , 9, 287 ( 1 9 7 2 ) .

13.

a)

Scully, Obstet.

J.A. McLachlan and R.L. Dixon, New C o n c e p t s i n S a f e t y E v a l u a t i o n , ( e d i t e d by M . A . Mewhlman, R.E. S h a p i r o and H. B l u m e n t h a l ) , Vol. 1, P a r t 1, p . 423, Hemisphere, Washington ( 1 9 7 6 ) .

HEXESTROL b)

373 M. M e t z l e r , R. G o l t s c h l i c h and J . A . Mc L a c h l a n , E s t r o g e n s i n t h e Environment, ( e d i t e d b y J . A . McLachlan), p. 293, Elsevier (1980).

14.

I . C a l a f e t e a n u , E. Dumitrescu and P. G r i n t e s c u , Revta Chim., 41, (1965); t h r o u g h Anal. A b s t r . 3786 (1966).

15.

261, ( 1 9 6 4 ) , I. K e r e n y l , Acta Pharma. hung., t h r o u g h Anal. A b s t r . 1 3 , 1980, ( 1 9 6 6 ) .

16.

V.G.

17.

The A d d i t i v e s i n Animal F e e d i n g - s t u f f s Subcommittee of t h e A n a l y t i c a l Methods c o m m i t t e e of t h e S o c i e t y f o r A n a l y t i c a l C h e m i s t r y . R e p o r t of t h e Hormones P a n e l . A n a l y s t , 88, 925, ( 1 9 6 3 ) .

18.

G,

13,

34,

B e l i k o v , Med. Prom. SSSR, 1 7 , 32, (1963).

M.J. Matherne, J r . , (1965).

J . A s s . o f f . a g r i c . chem.,

48, 613,

g,

19.

L. I. Kovdlenko, F a r m a t s i y a , 5 4 , (1969) ; t h r o u g h Chem. A b s t r . 7 2 , 59121e, (1970).

20.

S.L.

Tompsett,

21.

S.L.

T o m p s e t t , J . Pharm. Pharmacol. 1 6 , 207,

22.

H. Van Waes, Rev. Agr. ( B r u s s e l s ) , 23, 1 1 3 5 , ( 1 9 7 0 ) ; t h r o u g h Chem. A b s t r . , 74, 632312, ( 1 9 7 1 ) .

23.

R. Verbeke, J . Chromatogr., 1 7 7 , 6 9 , (1979); t h r o u g h Chem. A b s t r . , 21, 206599q, (1979).

24.

H. J . S t a n and F.Y. H o h l s , Z. Lebensm. - Unters F o r s c h . , 1 6 6 , 257, (1978); t h r o u g h Anal. A b s t r . 35, 6F16, (1978).

25.

J. Fharm. Pharmacol. 13, 747, ( 1 9 6 1 ) . (1964).

.

H. Jare, 0. R u t t n e r , W. Kroeza, F l e i s c h w i r t s c h a f t , 86, 3 6 3 1 t , (1977).

56, 1326, ( 1 9 7 6 ) ; t h r o u g h Chem. A b s t r . ,

I . Karkocha, Rocz. Fanstw. Z a k l . H i g . , 26, 497, (1975); t h r o u g h Chem. A b s t r . , 8 4 , ?368w, ( 1 9 7 6 ) . R. F e r r a n d o and A. R e n a r d , B u l l . SOC. Chim. b i o l . , 50, 1855 ( 1 9 6 8 ) .

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314

28. 29.

T. L a r s , J . Chromatgr.,

48,

560, (1970).

H. J a r c , 0. R u t t n e r , and \J. Krocza, J . Chromatogr.,

1 3 4 , 351, (1977). 30.

31.

H.C. H s i n , J a p a n A n a l y s t , 23, 1 2 2 6 , (1974); t h r o u g h Anal. A b s t r . 29, 2E16, (1975).

A.V.

J a i n , E.D.

S c h o l l , J . Assoc. O f f . Anal. Chem.,

58, 818, (1975).

130,

32.

F . J . Lawrence and J . J . Ryan, J . Chromatogr., 97, (1977).

33.

H. Ehrsson, T. Walle and H. B r o e t e l l , A c t a . Pharm. Suec., 8 , 319, (1971).

34.

M. G a b r i e l l a . C. Guido and S. P a t r i z i a , A n n a l i 1st. Sup. Sanita, 5, 586, ( 1 9 6 9 ) ; t h r o u g h Anal. A b s t r . , 2051; (1971).

35.

P . J . Cooper, M.J. d e F a u b e r t Maunder and G . J . McCutcheon, A n a l y s t , 92, 382, ( 1 9 6 7 ) .

36.

C.G.B. F r i s c h k o r n , M.R. Smyth, H.E. F r i s c h k o r n and J . Golimowski. F r e s e n i u s ' Z. Anal. Chem. 300, 407 (1980).

37.

M.R. Smyth, C.G.B. F r i s c h k o r n , F r e s e n i u s ' 2. Anal. Chem. 301, 220 (1980).

38.

G . H o e l l e r e r and D. J a h r , Z. L e b e n s m i t t e l u n t e r s .

39.

H.H. H e i n e r t , Arch. L e b e n s m i t t e l h y g . , 26, 1 5 2 , ( 1 9 7 5 ) ; t h r o u g h Chem. A b s t r . 8 3 , 162274h, (1975).

40.

D.H. Liem, L.G. H u i s i n ' t Veld, G . J . R u n d e r v o o r t , J. R o o s e l a a r , J. Ten Have, J. SOC. Cosmet. Chem., 307 (1976); t h r o u g h Chem. A b s t r . , 8 8 , 41529q (1978).

20,

U. - F o r s c h . , 157, 65, ( 1 9 7 5 ) ; t h r o u g h Anal. A b s t r . 29, - 4 D 83, (1975).

27,

MESTRANOL Hum&

1.

2.

3. 4. 5.

A . El-Obeid and Abdulluh A. Al-Badr

376 316 316 377 377 371 377 377 377 311 311 378 385 387 390 390 391 391 404

Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance Physical Properties 2.1 Crystal Data 2.2 Melting Point 2.3 Solubility 2.4 Identification 2.5 Spectral Properties Synthesis Absorption, Metabolism, and Excretion Methods of Analysis 5.1 Titrimetric Methods 5.2 Spectrophotometric Methoas 5.3 ChromatographicMethods References

Analytical Profiles 01Drug Subslancea Volume I I

375

Copyright 01982 by The American Pharmaceutical Association ISBN 0-12-260811-9

376

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

1. Description

1.1 Nomenclature 1.11 Chemical Names Ethinyloestradiol-3-methyl ether. 1 7 a-Ethnylestradiol-3-methyl ether 3-Plethoxy-19-nor-17 a-pregna-1,3,5(10)-trien-20yn-17-01. 19-Norpregna-1,3,5(1O)-trien-2O-yn-l7-ol,3methoxy- (17a)17a-Ethynyl-3-methoxy-1,3,5(lO)-estratrien-l7~-01. 17~-Ethynyl-3-methoxyoestra-ly3,5(lO)-trien-l7-ol. 17~t-Ethynyl-1,3,5(10)-estratriene-3, 17B-diol-3methyl ether. 1.12 Generic Name Mestranol. 1.13 Trade Names Mestranol is an ingredient of the following propietary oral contraceptive preparations: Conovid, C-Quens, Enavid, Enovid, Metrulen, Norinyl, Previson, Ortho-Novum, Ovanon, Ovulen, Sequens, Syntex Menophase. 1 . 2 Formulae

1.21 Empirical C21H2602 1 . 2 2 Structural

OH

377

MESTRANOL

1.23 CAS No. 72-33-3 1.3 Molecular Weight 310.42

1.4 Elemental Composition C 81.25%, H 8.44%, 0 10.31%. 1.5 Appearance White crystalline powder. 2. Physical Properties 2.1 Crystal Data Crystal data were reported by Ohrt et a1 (1) for some esterone-related compounds. The data given for mestranol was : a 6.998, b 39.737, c 6.8718., B117.58', Pz1, 2 = 4. 2.2 Melting Point Melts between 146' and 1 5 4 O with a range of 4'(2,3). 2.3 Solubility Almost insoluble in water, soluble 1 in 44 of ethanol, 1 in 23 of ether, and 1 in 4.5 of chloroform, 1 in 12 of dioxane, 1 in 23 of acetone, slightly soluble in methanol ( 2 , 4 , 5 ) . 2.4 Identification 2.41 Infrared Spectroscopic Test B.P. (4) and U.S.P. XIX (2) make use of the infrared absorption spectrum of mestranol as me n of identification of the drug. The infrared absorption spectrum of the sample exhibits maxima which are only at the same wavelengths as, and have similar relative intensities to, those in the spectrum of a standard mestranol. The infrared spectrum of mestranol will be discussed later

318

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

in the spectral properties of the drug. 2.42 Ultraviolet Spectroscopic Test

U.S.P. X I X requires that for the identification o f the drug, the ultraviolet absorption spectrum of methanolic solution exhibits maxima and minima at the same wavelengths as that of a similar solution of a standard mestranol, concomitantly measured. The ultraviolet spectrum of mestranol will be discussed later in the spectral properties of the drug. 2.43 Thin Layer Chromatographic Test

B.P. and U.S.P. XIX describe a thin layer chromatographic method for the identification of mestranol in which the principal spot in the chromatogram of the substance being examined is compared with that of mestranol obtained under identical conditions. 2.44 Color Test

According to B.P. a solution of mestranol in sulfuric acid appears orange-red by transmitted light, shows a yellowish-green fluorescence by reflected light and produces a reddish-brown f l o cculent precipitate after addition of ferric ammonium sulfate solution and a rose-red flocculent precipitate when water is added. 2.45 Solubility Test

B.P. uses a solubility test to distinguish mestranol from ethinylestradiol. The former is insoluble in a 5 % wfv solution of potassium hydroxide. 2.5 Spectral Properties 2 . 5 1 Ultraviolet Spectrum

The ultraviolet absorption spectrum of mestranol obtained from a solution in neutral methanol in the region of 200 to 350 nm using a Varian Cary 219 spectrophotometer is shown in Figure I. Two absorption maxima at about 2 1 8 and 286 nm and

379

MESTRANOL

J c c CI

F i g . 1. methanol.

U l t r a v i o l e t spectrum of m e s t r a n o l i n n e u t r a l

380

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

two minima at about 246 and 284 nm were observed. Reported (5) ultraviolet absorption spectrum of mestranol in methanol exhibited two maxima at 279 nm (E 1%, 1 cm 82) and 287.5 nm (E 1%,lcm 14.4). 2 . 5 2 Infrared Spectrum

The infrared spectrum of mestranol is presented in Figure 2 . The spectrum was obtained from nujol mull using a Unicam SP 3-300 infrared spectrophotometer. The spectral assignments are presented in Table 1. Table 1. Infrared spectral assignments for mestranol. Band Frequencyl Wavenumber cm

Structural assignment

1460

0-H stretching. ECH stretch. -C=C-stretch of the aromatic ring. C-H Deformation of -CH2-

1380

-C-H Deformation of -C-CH

1300 65 3

C-0-C stretch. 32-H Deformation.

3500 3 300 1510, 1620

3

2 . 5 3 Proton Magnetic Resonance (PIR) Spectrum.

The PMR spectrum of mestranol is presented in Figure 3 . The sample was dissolved in CDCl and 3

the spectrum obtained on a Varian-T60A NMR spectrometer with tetramethylsilane as the internal standard. The spectral assignments shown in Table 2 , are in agreement with reported studies (6) *

W.Vd#@P-

3

4

5

5

6

7

9

10

11

12

13 (4I S U

100

w

u 7u

60 50

44

34 20 10 0

Fig. 2.

I n f r a r e d spectrum of mestranol; Nujol M u l l .

do0

200 axyl -H

ti

F i g . 3. PMR spectrum of m e s t r a n o l i n CDC13 w i t h TMS i n t e r n a l standard

0 wa

MESTRANOL

383

Table 2.

PMR spectral assignments for mestranol.

Chemical Shift. PPd6)

Multiplicity Number of Protons.

Species

0.92

singlet

3

-CH3

2.12

singlet

1

-OH

2.55

singlet

1

acetylenic proton.

3.72

singlet

3

methoxy protons.

6.87

multiplet.

3

Aromatic protons.

2.54 I3C - Nuclear Magnetic Resonance (13C NMR) Spectrum The I3C NMR spectrum of mestranol in CDCl using 3 tetramethylsilane as an internal standard reference was obtained on a Jeol FX 100, 100 MHz instrument at an ambient temperature using a lorn. sample tube. The spectrum is presented in Figure 4 and the chemical shift values, derived from the off-resonance spectrum, is shown in Table 3. Table 3. I3C NMR spectral assignments for mestranol. Carbon

Chemical shift.

1 2 3 4 5 10

Carbon No.

No.

126.2590 113.9788 157.5908 111.5932 137.9037 132.6407

Chemical shift. (ppm)

13 17 18 20 21 CH3-0-

47.2199 79.9181 12.7185 87.7616 73.9221 55.2115

2.55 Mass spectrum and Fragmentometry The mass spectrum of mestranol, obtained by electron impact ionization, using Nermag GC-Mass spectrometer model R 1010, is presented in Figure 5. The spectrum shows a molecular ion PI? at m/e 310 (relative intensity 24.4%) and a base peak

384

J 1 3 C NMR spectrum of mestranol i n CDC13 with TMS Fig. 4. i n t e r n a l reference.

11

Fig.

5.

Mass spectrum of mestranol (EI).

385

MESTRANOL

at mfe 227. Based on the data interpretation and summary (INTSIJM) program presented by Smith et al, (7) for the possible fragmentation of the basic skeleton of estrogenic steroids, a proposed fragmentation pattern of mestranol is shown in Table 4. Table 4 . mle -

Proposed mass fragmentation pattern of mestranol. Relative intensity %

ion -

311

6.3

M+I

310

24.4

M+

242

15.7

C16H18d

228

19

‘1bH20q

227

100

160

10

159

14.6

147

20.5

145

11.9

129

20.7

128

26.8

116

13.9

+ +

C15H150? -t

Cl0H93 +

3. Synthesis Mestranol was prepared by Colton et al. (8) as f o l l o w s : Estrone {I} i s converted to its 3-methoxy analog (11) by reaction with methyl sulfate. The ethynyl group may then be introduced at position 1 7 either through reaction with sodium acetylide in liquid ammonia followed by hydrolysis o f the sodoxy compound, or through Grignardization with ethynyl magnesium bromide. Almost the sole product of the ethynylation reaction is that which results from attack of reagent from the least hindered a-side of the steroid,Fig.6.

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

3 86

3

t I11

J

0

HCEC N&iq

.NH3

&’ &Br

TECH

CH30

&CH

t

CH30

Figure 6.

Synthesis of mestranol.

ONa

MESTRANOL

387

4. Absorption, Metabolism and Excretion Mestranol is a synthetic estrogen which is more potent than estradiol. It is readily absorbed from the gastrointestinal tract and is slowly metabolized and excreted in urine and feces. The absorption, metabolism and excretion of the drug has been extensively studied in animals and humans I Fig. 7. Wijmenga and Van der Molen (9) reported a biological halflife of mestranol of 50 hours and that a small proportion of the drug was excreted in milk of nursing mothers. Mills et al, (10) showed that after the injection of tritium-labelled mestranol to women, the radioactivity representing the metabolites of the drug in the blood disappeared with an average half-life of 45 hours (range et al, (11) studied the metabolic 37-65 hours). Mahesh clearance rate and blood half-life of mestranol. The metabolic clearance rate of the drug after a single i.v. injection to women was 1265Llday. The radioactivity halflife after injection was 45.1 hours. No change in the metabolic clearance rate of the drug was noticed for upto 7 months of oral contraceptive medication. Using the constant infusion technique, Bird & Clark (12) measured the metabolic clearance rate of mestranol in normal young women and found it to be 1741L124 hours. The mean conversion ratio for mestranol to ethynylestradiol was 0.236 and to ethynylestradiol sulfate 3.369; that of mestranol to the sulfate was 6.476. The mean transfer constant for mestranol to ethynylestradiol was 0.182. The principal circulating form of the drug was ethynylestradiol sulfate. Mills et a1 (13) administered mestranol orally to normal women in order to measure the metabolic clearance rate and to study the urinary excretion of the drug. Mestranol was rapidly cleared from the plasma with an average metabolic clearance rate of 1247Llday. About 30% of mestranol administered was excreted in urine in 5 days, less than 4% in an unconjugated form, 10% as sulfate conjugates and about 52 as glucuronide conjugates. Comparison of the metabolic clearance and urinary excretion rates made before and again after six cycles of treatment with OrthoNovum SQ containing mestranol showed that prolonged administration had no effect on the metabolic clearance rate, rate of urinary excretion or mode of conjugation of the drug or its metabolites.

388

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

In a study by Bolt and Remmer (14) following the i.v. administration of 14C-Labelled mestranol to female rats; 45 and 3% of the radioactivity were found to be excreted in the feces and urine, respectively, in 3 days. Only 2 . 5 % of the radioactivity was expired as 14CO2 within 4 days indicating no significant degradation of the steroid nucleus. Some metabolites separated from fecal extracts had an unaltered 3-methoxy group. Others, however, were demethylated to derivatives of ethynylestradiol. The chronic i.v. injection of radioactive mestranol into mice resulted, as reported by Bolt and Remmer ( 1 5 ) , in an accumulation of radioactivity in organs to a greater extent than activity accumulated after radioactive estradiol administration. The metabolites of mestranol were tightly bound to the liver tissue and were not removed by solvent extraction or by acid hydrolysis. The high demethylation rate of mestranol in mice as compared to that in rats may be due to a high activity of the microsomal oxidase in the mouse liver. Following a single i.v. injection of the steroid into female rats, 1.5% of the radioactivity was recovered from urine collected in the first 3 days, and 55% of the activity was excreted in the feces within 3 weeks. Hanasono and Fischer (16) studied the excretion of tritiumlabelled mestranol and other contraceptive steriods and the enterohepatic circulation (EHC) of their metabolites in female rats. Mestranol was rapidly and extensively eliminated as metabolites in the bile after a single i.v. dose. The cumulative percentage of administered radioactivity appearing in the bile at the end of 8 hours was 6 9 % . Mestranol appeared in the bile primarily as a glucuronide conjugate and other polar materials which were not sulfate conjugates. Intact female rats given single i.p. doses of the labelled steroid eliminated radioactive metabolites in the urine and feces at a much slower rate than that seen in the bile of animals with biliary fistulas. Fecal excretion was the major route of elimination in intact animals for the steroid and accounted for 80% or more of the radioactive dose by the end of 7 days. Experiments were also conducted to assess the enterohepatic circulation of metabolites of the steroid. 59% of the intraduodenally infused radioactivity associated with the biliary metabolites of mestranol, underwent enterohepatic circulation and appeared in the bile during a 24 hour period. The glucuronide conjugate fraction of biliary metabolites was the most important fraction undergoing enterohepatic circulation. The

389

MESTRANOL

T

Glucuronide and sulfate conjugates

17a-Ethynylestradiol

H O HO

@PCH 1 2-Hydroxy-17a-ethynylestradiol.

OH

0

17a-D-Homoestradiol Figure 7.

D-Homoestrone

Metabolism of mestranol.

390

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

polar metabolites, which could not be hydrolysed by 6-glucuronidase, were not absorbed from the intestine and re-excreted in the bile. The authors conclude that the enterohepatic circulation is an important feature of the disposition of the contraceptive steroid in rats and is dependent on the excretion of glucuronide conjugates of the steroid metabolites in the bile. In a study using radioactive mestranol, Williams (17) identified 17 a-ethynylestradiol as a urinary metabolite using reverse isotope dilution technique. Abdel-Aziz and Williams (18), studying the urinary metabolites of mestranol in guinea pigs, identified 17 a-ethynylesteradiol conjugates, D-homoestradiol-176 and D-homoestrone, as metabolites. 3

14

In their study of the metabolism of 4- H and 4- C-mestraet a1 ( 1 9 ) , reported that reactions no1 by women, Williams involving position 4 were no greater than 1.7 to 3% of the dose as measured by the liberation of 3H into body water. The extent of deethynylation in vivo was no greater than 1 to 2% of the dose as measured by urinary estrone metabolites. Mestranol (0.7 and 0.32% of the dose), 17a-ethynylestradiol ( 6 . 6 and 11.3%) and 2-hydroxy-17a-ethynylestradiol ( 0 . 6 4 and 0.7%) were identified as metabolite glycons by reverse isotope dilution after ketodase hydrolysis of the urine from two of the women.

5. Methods of Analysis 5.1 Titrimetric Methods

(a)

The B.P. ( 4 ) recommended a titrimetric method in which the drug in tetrahydrofuran is treated with silver nitrate and then titrated with standard sodium hydroxide solution. The end point determined potentiometrically.

(b)

An argentimetric determination of some acetylene steroidal drugs, including mestranol, had been et _ a1 (20). The assay method developed by Roushdi _ depends on the precipitation of the steroid silver salt from alcoholic ammoniacal silver nitrate solution followed by extraction with a suitable organic solvent, evaporation of the solvent and then applying Volhard's method.

MESTRANOL

(c)

391

Roushdi et a1 (21) used a nonaqueous titrimetric method for determination of acetylenic steroids. The method involves the precipitation of the steroid silver salt using ammoniacal silver nitrate solution. The salts extracted will chloroform or ether and titrated with perchloric acid in acetic acid using Gentian Violet as indicator.

5.2 Spectrophotometric Methods 5.21 Ultraviolet Spectrometric Methods a) Bastow (22) deternined mestranol from its absorption at 280 nm after elimination of the interfering ketonic absorption by reduction with borohydride. Residual interference was allowed for by a three point correction. b) Gorog and Csizer (23) described methods for the determination of (i) mestranol as an impurity in norethynodreal and norethisterone and (ii) mestranol in contraceptive tablets. For (i) the sample is dissolved in methanol and treated with sodium borohydride. A solution of mestrano1 is treated sinilarly. The extinctions of the two solutions are measured at 287, 290.5 and 294 nm, and the mestranol content is calculated from a given equation. For (ii) the powdered tablets are heated under reflux with dichloromethane. After filtration and evaporation of the solvent, mestranol is determined as in (i). The mean recovery for the latter method was 98.3 ? 2.3%. c) A specific spectrophotometric method for the determination of 17-ethynyl steroids was published by Szepesi and Gorog (24). 17a-Ethynyl steroids were determined by quantitative conversion to 17-0x0 steroids by sodium t-butoxide at 81°, followed by spectrometric determination of the 16-glyoxalyl derivatives, Anlax. 294 nm, E 10,700, standard deviation 1.1%. 0 By the same principle, at 0 , the 17-0x0 impurity content in 17-ethynyl steroids was determined. The method was not applicable to +OX0 steroids.

392

HUMEIDA A. EL-OBEID AND ABDULLAH A. ALBADR

d) Shroff and Grodsky ( 2 5 ) described an ultraviolet spectrophotometric method for the estimation of mestranol in fresh tablets. In this method the sample is shaken with water and methylcyclohexane, centrifuged and the extinction of the upper phase measured at 287.7 nm. Baseline corrections applied are calculated from (i) the extinction at 302 nm and (ii) the extinctions at 278 nm and 302 nm and the extincfor pure mestranol; tion ratio E to E 278 287.7 for the the two corrected values of E287. 7

sample should agree within 2 0.006%. The contents of mestranol is calculated from the mean corrected value. The coefficient of variation for ten samples is 1.14%. The method is not preferred for aged tablets because on storage an interfering complex is formed between poly (vinyl pyrrolidone) and magnesium stearate excipients. 5.22 Colorimetric Methods

a) Chlorine o-tolidine reagent was used by Huettenrauch ( 2 6 ) for the selective detection of steroids with aromatic ring A and C13-OH group whether this was free, esterified or ether-linked. Mestranol gave positive reaction with sensitivity of 1-2 pg/cc.

b) Shroff and Huettemann ( 2 7 ) developed a colorimetric method for assaying mestranol in tablets. The method is based on the formation of a colored complex with phenol-sulfuric acid reagent. This complex exhibits an absorption maximum of 550 nm, obeys Beer's Law and is stable for reasonable length of time. According to the method the tablets are moistened with water and shaken with methylcyclohoxane. An aliquot of the organic phase is evaporated in an atmosphere of nitrogen and treated with phenol-sulfuric acid reagent and the extinction measured at 5 5 0 nm against a reagent blank. The error and precision are 2 1 . 6 % and 1 . 5 2 % respectively. The presence of norethisterone and excipients do not interfere.

393

MESTRANOL

The method is claimed to be superior to the ultraviolet spectrophotometric method described by Shroff and Grodsky and summarized above. c) A direct colorimetric method for the assay of mestranol in one tablet is reported by Comer et a1 (28). In this method one tablet is dissolved in sulfuric acidlmethanol (7:3) and the extinction measured at 545 nm against a reagent blank. To avoid interference by chlormadinone, the temperature must be kept below The coefficient 5 O during the color reaction. of variation was 1 . 0 5 % . Very similar methods have also been described by Templeton et a1 (29) for one tablet assay and by Wu (30) for mestranol in combination with ethynodiol diacetate. The latter procedure require prior chromatographic separation of mestranol.

d) Beyer (31) developed an automated colorimetric procedure for the quantitation of mestranol in tablets according to which the tablets are suspended in water and extracted with chloroformethanol. In an automatic system, re-extraction is effected with 10% ethanol solution in 90% sulfuric acid. The extinction is measured at 538 nm. The coefficient of variation is 21%. e) Rizk et a1 (32) determined mestranol colorimetrically by means of silver ions. An ethanolic solution is treated with aqueous ammonia and an excess of aqueous silver nitrate and the precipitated silver compound is filtered off. Excess silver ions in the filterate is determined by a dithizone spectrophotometric procedure and the amount of steroid is calculated from the silver ions consumed. 5 . 2 3 Infrared Spectroscopic Methods

a) Mestranol was determined in chloroform solution -1 by infrared spectrophotometry (33) at 3308 cm -1 (aromatic (Ethynyl vibrations) or 1 5 0 2 cm system vibrations).

394

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

b) Chatten et a1 (34) reported a quantitative assay of antifertility agents by infrared spectroscopy. Two pure estrogenic substances and eight pure progestogens were analysed in the selected wavelength range and shown to obey Beer-Lambert Law. Although progestogen analysis in compound tablets was accomplished in several cases using the technique, the estrogenic content in all compound tablets was too low t o permit analysis. c) Beyermann and Roder (35) analysed mestranol in oral contraceptives by infrared spectrophotometry after separation of the components by thin-layer chromatography. The separation was performed on silica gel with ethyl acetate as a solvent. The appropriate spot extracted with chloroform and the infrared spectrum in liquid paraffin mull was recorded and mestranol determined from its extinction at 7.95 um. 5 . 2 4 Spectrofluorometric Methods

a) Cullen et a1 ( 3 6 ) developed a sensitive procedure for the analysis of norgestrel and structurally related steriods based on sulfuric acid induced fluorescence. The selectivity of the reaction and mechanism of fluorescence formation were studied. The reaction is specific for A4 -3-ketosteroids which have both a 17Bhydroxy and a 17a-alkyl or alkyne substitution and A 1y3y5(10)-triene-3-01 steroids. A twostep mechanism was tentatively explained on the basis of the effects of temperature, time, initial acid concentration and subsequent dilution with water on fluorogen development. The procedure has been automated to permit unit dose analysis. b) A spectrofluorometric procedure for the determination of mestranol in some oral contraceptive tablets had been developed (37) which utilizes the native fluorescence of mestranol. The method is claimed to be rapid, reliable and sensitive. No separations are required and the method is applicable in the presence of norethynodrel, ethynodiol diacetate and norethisterone,

MESTRANOL

395

but chlormadinone acetate interferes. The method is applicable to single-tablet analysis. According to the procedure one powdered tablet is shaken with anhydrous ethanol and centrifuged. An aliquot of the supernatant is diluted with ethanol and the fluorescence at 327 nm (excitation at 284 nm) is compared with that of a standard solution. Anhydrous ethanol is used as a reference solution and a correction applied for its fluorescence. The accuracy under the conditions studied was 2 1.29% and the precision ranged from 2 0.896 to ?r 1.73. c) Mariani and Mariani-Vicari (38) also described a method for the assay of mestranol spectrofluorometrically. A sulfuric acid-methanol (1:l) reagent is used and a previous separation of the two components (ethynylestradiol and mestranol) in the compound preparation is not required. For tablets the hydrolysis of the starch by diastase is followed by extraction into chloroform. The wavelengths of emission and excitation spectra are 515 nm and 475 nm respectively. d) An automated fluorimetric method for one-tablet assay of mestranol was carried out by Comer et a1 (28). One tablet i s dissolved in sulfuric acid-methanol (7:3) and the fluorescence measured with Kodak Wratten N0.58 ,& Corning 1-60 primary filters and a Kodak Wratten 22 secondary filter. A scheme for automation of the fluorimetric method is illustrated. In twelve replicate experiments, the coefficient of variation was 1.30%. The fluorescence reaction is retarded by NO NO2 and H 0 Related ster3’ 2 2’ oids, such as estradiol methyl ether, esterone methyl ether and esterone, cause little interference.

e) Templeton et a1 ( 2 9 ) described a fluorimetric assay method of mestranol in which one tablet is allowed to disintegrate in 20% sodium hydroxide solution and water, then extracted with chloroform and an aliquot of the extract evaporated to dryness. The residue dissolved in 50% methanol solution in concentrated sulfuric acid and the fluorescence measured at 498 nm, with excitation at 468 nm.

396

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR f> A fluorimetric

assay method for the determination of mixture of mestranol and norethynodrel was reported by Pastor et a1 (39). For mestrano1 alone extracts (in 1 : 5 chloroform-methanol) were evaported to dryness, dissolved in ethanol cooled, and allowed to react with a mixture of 1 : 3 acetic acid-sulfuric acid at room temperature before measuring the fluorescence emission at 560 nm after excitation at 545 nm. When norethynodrel was present with mestranol, the extracts were treated with borohydride in methanol at room temperature before addition of concentrated hydrochloric acid and processing as above to estimate mestranol. Norethynodrel was estimated in the mixture, from borohydride and acetic acid-sulfuric acid treatment, by excitation at 485 nm and emission at 520 nm. Variation in measurements were 3% for mestranol and 2% for norethynodrel.

g) Mestranol in oral contraceptive tablets was also determined fluorirnetrically ( 4 0 ) by disintegrating a tablet in dilute hydrochloric acid, extracting with methylene chloride, treating with 0.2% hydroquinone (in 70:30 sulfuric acidethanol) and measuring the fluorescence.

h) Dusinsky and Radejova (41) reported fluorimetric methods for the determination of mestranol and some other estrogenic hormones. Methods, based on the determination of 'native' fluorescence and that 'induced' by sulfuric acid, for determining mestranol, ethinylestradiol, estradiol benzoate and valerate and the total estrogenic hormone content in mixtures of natural conjugated estrogens are described. The methods are claimed to be simple, highly sensitive and rapid. There is no need €or preliminary clean-up or hydrolysis. 5.25 Nuclear Magnetic Resonance (NMR) Spectrometric

Methods

Avdvich et al(6) used an NMR technique to quantitate mestranol bulk drug. Diphenylacetic acid was used as the internal standard and pyridine as the solvent. The amount of mestranol was calculated from the integrals of the peaks at 6.33 ppm

MESTRANOL

397

(methoxyl protons) and at 6.80 ppm (ethynyl proton de-shielded by pyridine). The average deviation was ? 0.6% and the results were in good agreement with those obtained by an official method. 5.3 Chromatographic Methods 5.31 Thin-Layer Chromatography

The literature describes several thin-layer chromatographic methods for the separation and analysis of mestranol in mixtures and contraceptive tablets. For quantitation, the plate is first scraped and mestranol eluted before treatment with a suitable reagent. Table 5 symmarizes thinlayer chromatographic methods for mestranol. 5.32 Column Chromatography a) Quantitative separation of progestins and es-

trogens from anovulatory formulations had been performed (57) using gel filteration on a synthetic polysaccharide (Sephade LH-20) and the compounds were determined directly by U.V. spectrophotometry.

b) Erunner and Kunze (58) published an analytical method for mestranol in combination with norethisterone (norethinadrone) or norethynodrel by partition chromatography and ultra-violet measurement. According to this procedure oral contraceptive tablets were extracted with dimethylformamide-formamide (1:l) and the steroids separated by partition chromatography with use of this solvent as stationary phase on Celite with heptane as mobile phase. Mestranol, in the relevant fraction, is determined by measuring the extinction at 287 nm with base-line correction obtained from the extinction at 302 and 315 nm. Interference was noticed with ethynodiol diacetate and chlormadinone acetate. The method had been adopted as official first action after a collaborative study was conducted (59). c) A method was described (60) for the separation of steroids in mixtures of pharmaceutical in-

terest by means of silicic acid column chro-

Table 5: Stationary Phase

Thin-Layer Chromatographic systems for mestranol analysis.

Developing solvent

Silica Gel or Neutral Allumina Kieselgel G

Silica Gel G/H

Cyclohexane/EtoAc (7:3) Ether/Cyclohexane

Visualization

Quantitative

0.5% Vanillin H2S04-EtON

-

-

Reference 42

Treat with 40% SbCl in measure extincgion 43 at 570 nm or fluoresence. HOAC

Iodine vabors

(8:2)

Treat with 15% TiCl in 3 RoAc-H2S04, measure extinction at 530 nm.

44

W

W

00

Propylene glycolimpregnated Kaieselguhr G.

Toluene, Cyclohexanetoluene ( 3 : 2 ) or petroleum ether.

Paraffin oil-impregnated -Kieselguhr G.

30,50 or 70% HoAC solutions.

Silica Gel

Benzenelethyl methyl ketone (9:l).

20% p-MeC H4S03H

-

45

-

45

in 947: E t h , heat at 120 for 10 min. Same as above.

-

Treat with H SO -i"ieOH(2:1)46 2 3 measure extinction at 540 run. contd......

Stationary Phase

Developing solvent

Visualization

Quantitative

Reference

EtoAc/cyclohexane/Me2C0 (5:1.5:2 )

85X PhosphoricMeOE (1:1) or SbCl -EoAc (1:l w/v>? heat at 12Oo-13O0. or Iodine vabors.

Use Icieselgel FIR 47,43 Treat with 15% TiCl conc. HC1. Pleasure 3 extinction.

Xieselgel G/HR a) 1-Dimensional

b) 2-Dimensional Above solvent followed by Cyclohexane/EtoAc (23:27). Silica Gel GF

2-Dirfiensional : Chloroform/methanol (9:l) H SO. and heat for 4 36 minutes at 1 00'. or Benzene/acetone(95:1) followed by benzene/ UV irradiation. methanol (95:5) or roethylene chloride/raethanol /water (150:9:0.5)

47,48

Visual comparison of 49 of the spots sizes and color intensities with standard spots.

-

Kiesel G

Benzene

Iodine vabors

Silica gel G

Heptane/acetone(4:1)

EtOII-conc. HCl(49 :1) Extract in propanol 51 plus H SO -H 0(7:3) 2 4 2 measure extinction at 545 nm.

Silica gel

-

HC1 gas and W irradiation.

50

-

52 contd...

....

Stationary phase

Developing solvent

Visualization

Silica gel G, A1 0 GF254 and 2 3 Silica gel GF 254

Petroleum ether/benzene/ He2C0 (5 :4 :1)

Iodine vabors, 2,4dinitro-phenylhydrazine or 2% H SO in 2 4 EtOIi.

Silica gel G-MiJ

Petroleum et'ner/?{e CO 2

50% H2S04

54

Silica gel 60 F254

2-Dimensional: Toluene-95Z/ethanol(9:1) followed by BuOAcfLight petroleum/AcOE(70:30:1)

H SO 2 4

55

Silica gel GF

BenzenefEtoAc (4:l)

0.5% H3 SeO -conc. H SO and Aeat at 04 100 for 15 min.

Quantitative

-

Reference

53

By spectrodensitome- 56 try by scanning at 360 nm by reference to an internal standard.

MESTRANOL

401

matography. Association of estrogenic with progestational steroids, in sone cases extended to androgens, were considered. The elution was performed with a gradient of ether in petroleum ether, obtained by means of two metering pumps. The effluent was monitored continuously by means of a hydrogen flame ionization detector, a part of the effluent being continuously drawn off t o the detector. The quantitative analysis of the steroids separated by the method was performed by a U.V. spectrophotometric or a colorimetric procedure. 5.33 Liquid Chromatography

Hara and Hayashi (61) studied the correlation of retention behaviour of steroidal pharmaceuticals in polar and bonded reverse-phase liquid column chromatography. For the systematization of correlation between the chemical structures of solutes and retention behaviour in liquid column chromatography, the retention volume of the modified steroids on silica (Corasil 11) and chemically bonded reverse-phase columns (Bondapak C18/Corasil) were studied using various binary solvent systems. Retention parameters for the functional groups of the steroids were calculated according to Martin's additive rule. By comparing these values obtained on normal and reverse-phase columns, characteristic features of both packings with regard to solute structures and solvent systems were elaborated. 5 . 3 4 Gas Chromatography

a) A comparison of an W spectrophotometric assay and a gas-liquid chromatographic assay was conducted (25) for mestranol in tablets. Both methods gave results in good agreement and both are suitable for fresh tablets. In the gaschromatographic method the column used was stainless steel (21 in x 0.25 in) of 4% of 0 XE-60 on Diatoport S, maintained at 195 Nitrogen was used as a carrier gas at 70-75 m l / hour. The method is preferred for the assay of aged tablets because on storage a complex between poly (vinylpyrolidone) and magnesium stearate excipients was produced which interfered with the W methods and was identified by

.

402

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

gas-liquid chromatography. b) Okuno and Higgins (62) reported a method for the determination of residues of mestranol and ethynylestradiol in foliage, soil and water samples. The lower limits for the detection of mestranol and its 3-hydroxy homolog, ethynylestradiol were 0.05, 0.1 and 0.01 ppm for foliage, soil and water respectively. Samples were extracted in an acid medium to free any conjugated ethynylestradiol and then cleaned up by Florisil column chromatography. Water samples were directly analysed by gas chromatography using a flame ionization detector and a column packed with OV 17 on Gas Chrom Q. Operating 0 temperatures were 260 for column, 275'for the inlet and 290' for the detector. Further cleaning up of the soil and vegetation samples was carried out by gel permeation and Florisil chromatography. Analysis was then carried out by gas chromatography. The lower limit of detection was lOOng for each steroid. c) Mestranol and norethisterone were determined in estrogen-progestin contraceptive tablets containing both components (63) by extraction with ethyl acetate and a single gas chromatography, with testosterone propionate as internal standard. The recoveries of mestranol and norethisterone were 98.54 and 99.14% respectively and the precision was 1.40 and 1.74% respectively. The procedure could be applied to single tablets containing 0.05 mg mestranol and 1 mg norethisterone.

d) Templeton et a1 (29) performed a gas-liquid chromatographic assay for mestranol. A tablet is allowed to disintegrate in sodium hydroxide in water, then extracted with chloroform. A dilute cholestane solution is used as an internal standard. The column used is a glass column (2 ft. x 4 mm) containing Diatoport S (80 to 100 mesh) impregnated with 6%0of silicone-gum rubber W-96 operated at 205 , with He (60 to 70 ml per min.) as carrier gas and flame ionization detector.

MESTRANOL

403 ACKNOWLEDGEMENT

The a u t h o r s wish t o t h a n k Mr. A l t a f Hussain Naqvi, f o r t y p i n g t h i s manuscript. A sample of m e s t r a n o l was k i n d l y donated by S e a r l e Research and Development, D i v i s i o n of G.D. S e a r l e and Co., Chicago, I l l i n o i s , USA.

404

HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR

REFERENCE 1. J.M. Ohrt, B.A. Haner and D.A. Norton, Acta Cryst., 1 7 ( 2 ) , 1 6 1 1 (1964). 2. The United-States Pharmacopeia, 19th ed., Mack Publishing Co., Easton, Pa., p. 1405 (1975). 3. Remington's Pharmaceutical Sciences, 15th ed., Mack Publishing Co., Easton, Pennsylvania p.920 (1975). 4 . British Pharmacopoeia, Her Majesty's Stationery Office, London, U.K., p. 291 (1973). 5 . E.G.C. Clarke, Isolation and Identification of Drugs, The Pharmaceutical Press, London, p . 4 0 5 (1969). 6 . E.W. Avdovich, M. Bowron and B.A. Lodge, J. Pharm. S c i . , 59(12), 1 8 2 1 (1970). 7. D.H. Smith, B.G. Buchanan, I?.C. White, E.A. Feigenbaum, J. Lederberg and C. Djerassi, Tetrahedron, 2, 3117 (1973). 8 . F.B. Cotton, L.N. Nysted, B. Reigel and A.L. Raynold, J. Amer. Chem. Soc., 79, 1 1 2 3 (1957). 9. H.G. Wijmenga and H.J. van der Molen, Acta endocr.,Copenh., 61, 665 (1969), per , 209, 2106 (1969). 10. T.M. Mills, T.J. Lin, W.E. Braselton, J.E. Ellegood and V.B. Mahesh, Am. J. Obstet. Gynecol., 1 2 6 ( 8 ) , 987 (1976). 11. V.B. Mahesh, T.M. Mills, T . J . Lin, J . O . Elegood and W. E. Braselton, Pharmacol. Steroid Contracept. Drugs, 117,30 (1977). 12. C.E. Bird and A.F. Clark, J. Clin, Endocrinol. Metab. 3 6 ( 2 ) , 296 (1973). 13. T.M. Mills, T.J. Lin, S . Hernandez-Ayub, R.B. Greenblatt, J . O . Ellegood and V.B. Mahesh, Am. J. Obstet. Gynecol., 1 2 0 ( 6 ) , 773 (1974). 1 4 . H.M. Bolt and H. Remmer, Xenbiotica, 20, 77 (1972). 1 5 . H.M. Bolt and H. Remmer, ibid, 2 ( 5 ) , 489 (1972). 1 6 . G.K. Hanasono and L.J. Fischer, Drug Hetab. Dispos., 2 ( 2 ) , 159 (1974). 1 7 . K . I . Williams, Steroids, 1 3 ( 4 ) , 539 (1969). 18. M . T . Abdel-Aziz and K . I . H . Williams, J . Drug Res., 60, 195 (1974). 1 9 . J.G. Williams, C. Longcope and K . I . H . Williams, Steroids, 2 5 ( 3 ) , 343 (1975). 20. I.M. Roushdi, A . I . El-Sebai and S . Belal, Egypt. J. Pharm. Sci., 1 5 ( 1 ) , 8 1 (1974). 21. I.M. Roushdi, A . I . El-Sebai and S. Belal, ibid, 1 5 ( 2 ) , 217 (1974). 22. R.A. Bastow, J. Pharm. Pharmacol., 1 9 ( 1 ) , 4 1 (1967). 23. S. Gorog and E. Csizer, Acta Chim. (Budapest), 6 5 ( 1 ) , 41(1970). 24. G. Szepesi and S. Gorog, Analyst, 9 9 ( 1 1 7 7 ) , 218 (1974).

MESTRANOL

405

25. A.P. S h r o f f and J . Grodsky, J . Pharm. S c i . , 5 6 ( 4 ) , 460 (1967). 26. R. H u e t t e n r a u c h , P h a r m a z i e , 2 0 ( 1 1 ) , 740 (1965). 27. A.P. S h r o f f a n d R.E. Huettemann, J . Pharm. S c i . , 5 6 ( 5 ) , 654 (1967). 28. J . P . Comer, P . Hartsaw a n d C.E. S t e v e n s o n , J . Pharm. S c i . , 5 7 ( 1 ) , 147 (1968). 29. R . J . Templeton, W.A. A r n e t t and I . M . J a k o v l j e v i c , 5 7 ( 7 ) , 1168 (1968). 30. J.Y.P. Wu, J . Assoc. O f f . Anal. Chem., 5 8 ( 1 ) , 75 (1975). 31. W.F. Beyer, J . Pharm. S c i . , 5 7 ( 8 ) , 1415 (1968). 32. M. R i z k , J.J. V a l l o n and A. Badinand, A n a l y t i c a chim.Acta, 6 5 ( 1 ) , 220 (1973). 33. E . Pawelczyk, I. P l u t a and B. M a r c i n i e c , Acta P o l . Pharm., 3 0 ( 3 ) , 289 (1973). 34. L,T.- C h a t t e n , E . J . T r i g g s and S . J . Glowach, J . Pharm. Belg. 3 1 ( 1 ) , 6 3 (1976). 35. K. Beyermann and E. Roder, Z. A n a l y t . Chem., 2 3 0 ( 5 ) , 347 (1967). 36. L . F . C u l l e n , J . G . R u t g e r s , P a . A. L u c c h e s i and G . J . Papar i e l l o , J . Pharm. S c i . , 5 7 ( 1 1 ) , 185 (1968). 37. J . Theron, J . Pharm. S c i . , 4 9 ( 1 1 ) , 1648 (1970). 38. A. M a r i a n i and C. Mariani-Vicari, Z e n t r a l b l . Pharm. Pharmakother. L a b o r a t o r u i m s d i a g n . , 111(1),1 9 (1972). 39. J . P a s t o r , A.M. P a u l i and N. P a p o c c h i a , Trav. SOC. Pharm. i v l o n t p e l l i e r , 3 3 ( 3 ) , 411 (1973). 40. J . H . M . Miller and P. Duguid, P r o c . Anal. Div. Chem. SOC., 1 3 ( 1 ) , 9 (1976). 41. G . Dusinsky and E. R a d e j o v a , Farm. Obz., 4 6 ( 5 ) , 225 (1977). 42. J . S . Hathews, Biochim. Biophys. Acta, 69, 1 6 3 (1963). 43. R. H u e t t e n r a u c h and I. K e i n e r , P h a r m a z z , 2 0 ( 4 ) , 242 (1965). 44. D. H e u s s e r , Dent. Apotheker-Ztg., 1 0 6 ( 1 3 ) , 4 1 1 (1966). 45. D. S o n a n i n i and L. Anker, Pharm. Acta Helv., 4 2 ( 1 ) 54 (1967). 46. P. Horak, Cesk. Farm., 1 6 ( 5 ) , 232 (1967). 47. E. Roder, Dent. Apotheker-Ztg., 1 0 7 ( 2 9 ) , 1007 (1967). 48. I. Szekacs a n d M. Klembala, Z . Klin. Chem. Klin. Biochem., 8 ( 2 ) , 1 3 1 (1970). 49. M.B. Simard and B.A. Lodge, J . Chromatogr., 5 1 ( 3 ) , 517 (1970). 50. H. Sachweh, P r o c . Conf. Appl. Phys. Chem., 2nd., 1,289 (1971). E d i t e d by Buzas, I l o n a . Akad. Kiado: B u d a p e s t ,

u,

51. 52. 53. 54.

Hung. G r o c h u l s k i , Acta P o l . Pharm., 2 8 ( 2 ) , 185 (1971). J e f f e r y , J . Chromatogr., 5 9 ( 1 ) , 216 (1971). Enache, Rev. Chim., 2 4 ( 5 ) , 648 (1973). M i j a t o v i c , L j . R i s t o v i c and J . G r u b o r , Arch. Farm.,

A. J. S. B.

2 3 ( 2 ) , 9 1 (1974).

406

HUMEIDA A. EL-OBEIDAND ABDULLAH A . AL-BADR

55. G. Cavina, G . Moretti and M. P e t r e l l a , J . Chromatogr., 1 0 3 ( 2 ) , 268 (1975). 56. A.P. Shroff and C . J . Shaw, J . Chromat. S c i . , 1 0 ( 8 ) , 509 (1972). 57. A. Alvarez Fernandez and N.V. T o r r e , J . Pharm. S c i . , 5 8 ( 6 ) , 740 (1969). 58. C.A. Brunner and F.M. Runze, J . A s s . O f f i c . Anal. Chem., 53(2). . . _ 234 (1970). 59. C.A. Brunner, J . A s s . O f f i c . Anal. Chem., 54(3), 590 (1971). 60. G . Cavina, G . M o r e t t i , A. M o l l i c a and R. A n t o n i n i , J., Chromatogr., 60 ( 2 ) , 1 7 9 (1971). 61. S. Hara and S. Hayashi, J. Chromatogr., 142, 689 (1977). 62. I. Okuno and W.H. H i g g i n s , B u l l . Environ. Contam. T o x i c o l . , 1 8 ( 4 ) , 428 (1977). 63. G. M o r e t t i , G . Cavina, G . C h i a p p e t t a , I. F a t t o r i , M. P e t r e l l a and V . Pompi, B u l l . Chim. Farm., 1 1 6 ( 8 ) , 463 (1977).

NOSCAPN Mohammed A. Al-Yahya and Mahmoud M . A . Hassan

408

1. Description 1. I Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste 2. Physical Properties 2.1 X-Ray Diffraction 2.2 Solubility 2.3 Dissociation Constant 2.4 Optical Rotation 2.5 Spectral Properties 3. Preparation 3.1 Isolation from Opium 4. Synthesis of Noscapine 4.1 Tissue Culture Method 4.2 Chemical Methods 5 . Biosynthesis of Noscapine 6. Metabolism 7. Methods of Analysis 7.1 Identification Tests 7.2 Microcrystal Tests 7.3 Titrimetric Methods 7.4 Complexometric Methods 7.5 Spectrophotometric Methods 7.6 Chromatographic Methods 8. References

Analytical Profilesof Drug Substances Volume 1 I

408

408 414 414 414 414 414 415 415 415 416 429 429 429 429 429 429 43 3 436 436 436 439 441 441 445 45 6

407

Copyight 0 1982 by The AmdUn phmn.ce~iiulAmciation ISBN 0-12-260811-9

408

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN

1. D e s c r i p t i o n 1.1

Nomenclature

1.1.1 Chemical Names a-

(S)-6,7-Dimethoxy-3-(5, 6, 7 , 8tetrahydro-4-methoxy-6-methyl-l , 3-d i o x o l o [ 4,5-g] i s o q u i n o l i n - 5 - y l ) - l (1) (3 H) -isobenzof uranone.

b-

1-a -2-methyl-8-methoxy-6,7-methylene dioxy-1- ( 6 , 7-dimethoxy-3-phthalidyl) 1, 2 , 3 , 4-tetra-hydroisoquinoline.

-

(1)*

1.1.2

c-

1 (3 H) Isobenzofuranone, 6 , 7 dimethoxy-3-(5, 6, 7 , 8-tetrahydro-4methoxy-6-methyl-1, 3-dioxolz-[4, 5-g] isoquinolin-5-y1)-, [s-(R* s ) I . (2)

d-

(3 S)-6, 7-dimethoxy-3-[(5R)-5, 6, 7 , 8-tetra-hydro-4-methoxy-6-methyl-1,3-dioxolo [ 4 , 5-g] isoquinolin-5-y1] phthalide. (3).

Generic Names d-Gnoscapine; 1-6-Narcot i n e ; I-Narcot i n e ; N a r c o t i n e ; Noscapine.

1.1.3

Trade Names Capval; Coscopin; Coscotabs; Keyt u s s c a p i n e ; Longatin; Lyobex; Fethoxyh y d r a s t i n e ; Narcompren; Narcosine; Marcotussin; N e i t a c l o n ; Nicolane; Nipaxon; Noscapal; Noscapalin; NSC 5366; Opian; Opianine; Terbenol; Tusscapine; Vadebex.

1.2

Formulae

1.2.1

Empirical

409

NOSCAPINE 1.2.2

Structural

OCH3

Nos c a p i n e The a l k a l o i d n o s c a p i n e can be c l e a v e d v e r y r e a d i l y i n t o two m o i e t i e s ; w i t h d i l u t e s u l f u r i c a c i d , c o t a r n i n e and o p i a n i c a c i d a r e g e n e r a t e d . Under a c i d i c r e d u c i n g c o n d i t i o n s , e.g., zinc i n hydrochloric acid o r s u l f u r i c a c i d , h y d r o c o t a r n i n e and meconine a r e formed (Scheme 1 ) . With t h e s t r u c t u r a l e l u c i d a t i o n of c o t a r n i n e , o p i a n i c a c i d , h y d r o c o t a r n i n e and meconine, and g i v e n t h e p r e s e n c e of a l a c t o n e r i n g i n n o s c a p i n e , the structure o f t h i s a l k a l o i d was e s s e n t i a l l y e s t a b l i s h e d ( 4 ) . 1.2.3

CAS No

(12 8- 62 - 1) 1.2.4

Niswesser L i n e N o t a t i o n T C566 DO FO K N EH & & T J H 01 K J T56 B VO D H J HOL 1 0 1 "ALPHA" LV.

1.2.5

S t e r e o c h e m i s t r y and A b s o l u t e C o n f i g u r a t i o n T h e s t e r e o c h e m i s t r y of n o s c a p i n e h a s been s t u d i e d by many w o r k e r s (5-9). The prolonged a c t i o n of h o t m e t h a n o l i c p o t a s s i u m h y d r o x i d e on n a t u r a l (-)- a - n a r c o t i n e r e s u l t s i n t h e f o r m a t i o n of a n e q u i l i b r i u m m i x t u r e of t h e o r i g i n a l b a s e and a new o p t i c a l l y

410

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN

'n'

'2

OCH3

Noscapine

OCH 3

OC!I

Hydrocotarnine

Cotarnine

+

+ CRO

OCH

OCH

Meco n ine

Opianic acid Scheme 1

41 1

NOSCAPINE a c t i v e d i a s t er eoisomer , (-) -6 -narc0 t i n e , which can be w r i t t e n a s shown i n Scheme 2. Lithium aluminum h y d r i d e r e d u c t i o n of t h e a- and 8-noscapines r e a d i l y a f f o r d s anarcotinediol and 6 - n a r c o t i n e d i o l respectively

.

1

2

A c e t y l a t i o n of t h e s e d i o l s g i v e s r i s e t o t h e corresponding d i a c e t a t e s 2 and k , b u t subsequent c a t a l y t i c h y d r o g e n o l y s i s y i e l d s one and t h e same d e x t r o r o t a t o r y benzyli s o q u i n o l i n e 2. The f o r e g o i n g sequence c l e a r l y e s t a b l i s h e s t h a t a- and 8-noscapine must d i f f e r from each o t h e r o n l y i n t h e i r s t e r e o c h e m i s t r y a t C-9. The b e n z y l i s o q u i n o l i n e 5 shows a p o s i t i v e Cotton e f f e c t near 295 m u , s o t h a t i t s C-1 hydrogen must b e a l p h a as i n d i c a t e d . I t f o l l o w s t h a t t h e C-1 hydrogen i n (-)-an a r c o t i n e and i n (-)- 6 - n a r c o t i n e must a l s o be a l p h a (Scheme 2 ) .

A 1 t e r n a t i v e l y , a-narco t ined i o l w a s c y c l i z ed v i a i t s monomesylate d e r i v a t i v e t o t h e Nm e t h o t e t ra h y d r o p r o t o b e r b e r i n e s a l t 5. T h i s material underwent N-demethylation on p y r o l y s i s t o y i e l d t h e p r o t o b e r b e r i n e b a s e 7. Reductive removal of t h e hydroxyl group w a s achieved i n e t h a n o l i c p e r c h l o r i c a c i d o v e r a palladium c a t a l y s t . The t e t r a h y d r o p r o t o b e r b e r i n e 8 t h u s o b t a i n e d showed a s t r o n g n e g a t i v e r o t a t i o n , so t h a t i t s C-14 hydrogen must be a l p h a . The i d e n t i c a l sequence w a s c a r r i e d o u t using p n a r c o t i n e d i o l t o y i e l d t h e tetrah y d r o p r o t o b e r b e r i n e b a s e ?. Hydrogenolytic c l e a v a g e of t h i s s p e c i e s t h e n provided t h e same 1evor o t a t o r y t e t r a hyd r o pro t o b er b er i n e 8. The c o n c l u s i o n i s t h a t t h e C-1 hydrogens i n b o t h a- and 6-noscapine a r e a l p h a (Scheme 3 ) . Turning t o t h e s t e r e o c h e m i s t r y a t C-9 f o r (-)-a and (-)- B-narcotine, m o l e c u l a r models i n d i c a t e d t h a t t h e d i h e d r a l a n g l e between t h e p r o t o n s a t C-13 and C-14 of t h e 13-a-hydroxy

-

412

MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN

CH3

OCH3 (-) -6-Narcotine

(-)-a-Narcotine ( n a t u r a l isomer)

1

1 --

L1A1H4

LiA1H4

OCH~ a-Narcotinediol

1.

1 -

B-Narcotinediol

1 --

2

-

-

Ac 20 , P Y ~

Ac 0, pyridine

idine

OAc CH20Ac OCH3 0CH3

Scheme 2

413

NOSCAPINE

H

........OH

pyridine

6 @CH3 d-Narcotinediol

7 -

as o u t l i n e d

9 OCH3 B-Narcotinediol Scheme 3

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN

414

b a s e 7 i s a b o u t 160'. On t h e o t h e r hand, f o r the 13-6 -hydroxy b a s e 2 d e r i v e d from 6 - n a r c o t i n e , t h i s a n g l e is o n l y a b o u t 60'. Following exchange of t h e h y d r o x y l i c p r o t o n s f o r d e u t e r i u m , i t w a s determined t h a t t h e s p l i t t i n g c o n s t a n t .J13,14 w a s 9 Hz f o r and o n l y a b o u t 1 . 5 Hz f o r 9. species The l a r g e c o u p l i n g v a l u e of 9 Hz i s accord w i t h a t r a n s arrangement of t h e C-13, 1 4 hydrogens i n I_, and t h e s m a l l c o u p l i n g c o n s t a n t of 1 . 5 Hz a r g u e s f o r a c i s r e l a t i o n s h i p i n 9, t h u s s e t t l i n g t h e s t e r e o c h e m i s t r y a t C-9 f& a- and 8-noscapine.

1,

1.3

iz

Molecular Weight 413.43

1.4

E l m e n t a l Composition C, 63.91%; H, 5.61%; N , 3.39%; 0 , 27.09%

1.5

Appearance, C o l o r , Odor

and Taste

Noscapine o c c u r s i n t h e form of Orthorhombic b i s p h e r o i d a l p r i s m s , t a b l e t s from d i a c e t o n e o r a s f i n e , almost w h i t e c r y s t a l l i n e powder. T r i b o l u m i n e s c e n t d 1.395. I t i s o d o r l e s s and tasteless. 2.

Physical Properties 2.1.1

X-ray d i f f r a c t i o n Crystallographic d a t a f o r noscopine are s c a r c e . The o n l y r e p o r t e d d a t a is due t o Love11 (10) and Steward and P l a y e r (11). These a r e a s f o l l o w s : Long needle-shaped c r y s t a l s were o b t a i n e d by r e c r y s t a l l i s a t i o n of t h e commercial n o s c a p i n e from e t h a n o l o r methanol. Weissenberg photographs t a k e n w i t h Cu Ka (1.5418 A)' r a d i a t i o n revealed the following systematic absences:

hOO, h

= 2n

+

1

OKO, K = 2n -I-1 001, 1 = 2n + 1

415

NOSCAPINE

d e f i n i n g unambiguously t h e space group P212121. C e l l dimensions were o b t a i n e d from 28 v a l u e s of 32 r e f l e x i o n s from n o s c a p i n e u s i n g two a x e s i n each c a s e , measured w i t h a counter diffractometer. The f o l l o w i n g d a t a were o b t a i n e d :

M.W.

413.41

178

M.p. (OC)

C r y s t a l system Space group Cell

Dimensions

(8)

d 3 )

r

P212121 15.398(12) b 32.686(36) c ( p r i m ) 8.022(8) 4037(11)

z

8

Qcalc (g. ~ m - ~ )

1.360

.

Qexp (g ~ m - ~ ) 2.1.2

O r t hor homb i c

1.38

Melting P o i n t 174-176OC(3) 176OC s u b l i m e s a t 15OoC-16O0C under 11 mm p r e s s u r e a t 2 mm d i s t a n c e (1)

2.2

Solubility I t i s i n s o l u b l e i n water; s l i g h t l y s o l u b l e i n a l c o h o l ( 9 5 % ) , i n e t h e r and i n carbon t e t r a c h l o r i d e . S o l u b l e i n chloroform, benzene and v e r y s o l u b l e i n a c e t o n e (12).

2.3

D i s s o c i a t i o n Constant I t i s a v e r y weak b a s e , pKa 7.8 ( 1 ) and 4.85 i n 80% m e t h y l c e l l o s o l v e ( 1 3 ) .

2.4

Optical Rotation

+

42'

to

+

(2% w/v i n 0 . 1 M h y d r o c h l o r i c a c i d ) (3) [ u I D - 198O (1% w/v i n c h l o r o f o r m ) ,

[a],

48'

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN

416

[a],

- 146 (2% w/v i n t o l u e n e ) , - 147' (1.59% i n b e n z e n e ) ,

[a],

+

[a],

2.5

50 (1% w/v i n h y d r o c h l o r i c a c i d ) (14)

Spectral Properties 2.5.1

U l t r a v i o l e t Spectrum The W spectrum of n o s c a p i n e i n methanol w a s scanned from 200 t o 400 nm u s i n g V a r i a n Carry 119 Spectrophotometer. I t e x h i b i t s a c h a r a c t e r i s t i c UV spectrum ( F i g . 1) w i t h two maxima: Xmax 310.2 290.6

1% cm 114.9 106.4

(C, 9.42 mg p e r 100 ml) (C, 9.42 mg p e r 1 0 0 ml)

Other UV s p e c t r a l d a t a of n o s c a p i n e have a l s o been r e p o r t e d : Xmax 209 291 309-310 Xmax 291 310 291 3 09 2.5.2

Log

E

4.86 3.60 3.69

) ) )

i n e t h a n o l (1, 15)

(E)

a b o u t 1.1 ) a b o u t 1.4 ) 3981 4898

i n a l c o h o l 95% (3)

1 )

i n methanol (12)

I n f r a r e d Spectrum The I R s p e c t r a of n o s c a p i n e a s K B r d i s c and n u j o l m u l l were r e c o r d e d on a P e r k i n E l m e r FT-680B s p e c t r o p h o t o m e t e r and shown in F i g . 2 t?, F i g . 3 respectively. The s t r u c t u r a l a s s i g n e m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s (Table 1)

417

NOSCAPINE

Fig.

1.

W Spectrum of N o s c a p i n e i n M e t h a n o l .

Wavelmgt h F *O

Fig.

2.

5.0

6.0

70

8.0

s.0

I R Spectrum of Noscapine as K B r d i s c .

f0

12

!4

1

2500

Fig. 3 .

.

2000

I R Spectrum of Noscapine as Nujol Mull.

800

700

420

MOHAMMED A . AL-YAHYA A N D MAHMOUD M. A . HASSAN T a b l e 1. I R C h a r a c t e r i s t i c s of Moscapine Frequency c m

-1

Assignement

3000, 2945, 2880, 2845, 2800

Methylened i o x y and C-H and -CH frequencies. 3 ( y - l a c t o n e ) 3-C=O g r o u p

17 60 1625 1600,1505,1480, 1280-1 22 5

-c=cAromatic Aromatic m e t h o x y - a r y l C-0 stretching vibrations.

790, 815, 8 3 5 , 885

2 a d j a c e n t H atoms, i s o l a t e d H atom C-H o u t of p l a n e d e f o r m a t i o n . Tetra and pen t a s u b s t i t u t e d b e n z e n e s

.

Other c h a r a c t e r i s t i c a b s o r p t i o n bands are: 1460, 1430, 1405, 1 3 9 0 , 1 3 8 0 , 1 3 6 5 , 1 3 3 0 , 1310, 1 2 0 0 , 1120, 1 0 8 5 , 1040, 1 0 1 0 , 980, 930, 900, B O O , 765, 750, 735, 725, 715, and 700 cm-'. O t h e r I R d a t a a r e a l s o r e p o r t e d (16) 2.5.3

N u c l e a r Magnetic Resonance S p e c t r a 2 . 5 . 3 . 1 P r o t o n Spectrum The PMR s p e c t r u m of n o s c a p i n e i n d e u t e r a t e d chloroform w a s r eco r d ed on a V a r i a n XL200, 2 0 0 MHz NMR s p e c t r o meter u s i n g t e t r a m e t h y l s i l a n e a s a r e f e r e n c e s t a n d a r d ( F i g . 4 ) . The following s t r u c t u r a l a s s i g n m e n t h a v e been made ( T a b l e 2 ) . 4

I, 0

7

F i g . 4.

6

5

4

PMR Spectrum of Noscapine and T e t r a m e t h y l s i l a n e

3

2

i n Deuterated Chloroform.

MOHAMMED A . AL-YAHYA AND MAHMOUD M.A. HASSAN

422

Table 2.

PMR C h a r a c t e r i s t i c s of Noscapine

Ass ignemen t (Group)

Po s i t i o n

Chemical S h i f t ( 6 )

3 , 4 of i s o q u i n o l i n e

2.32 (m)

N- CH3

2 of i s o q u i n o l i n e

2.53 ( s )

OCH3

8 of i s o q u i n o l i n e

3.84 ( s )

OCH3

&'of p h t h a l i d y l

4.02 (s)

OCH3

5'of

4.08 ( s )

-CH2-CH2

phthalidyl

-CH-

1 of i s o q u i n o l i n e

4.37 (d)

-CH-

9 of p h t h a l i d y l

5.55 (d)

-CH2-

methylened i o x y

5.92 ( s )

-CH-

2'0f

6.05 (d)

-CH-

5 of i s o q u i n o l i n e

6.29 ( s )

-CH-

3'of

6.94 (d)

phthalidyl

phthalidyl

s = s i n g l e t , d = doublet, m = multiplet

Other PMR s p e c t r a l d a t a was a l s o r e p o r t e d ( 1 7 an.d 5 5 ) . 2.5.3.2

I3C-NMR

Spectra

I3C-NMR c o m p l e t e l y decoupled and o f f - r e s o n a n c e s p e c t r a are shown i n Fig. 5 and Fig. 6 r e s p e c t i v e l y . Both were r e c o r d e d o v e r 11001.1 HZ r a n g e , i n d e u t e r a t e d c h l o r o f o r m (CDC13) on XL-200,200 MHz NMR s p e c t r o m e t e r . Using 10 mm sample t u b e and tetramethylsilane as reference standard a t 25OC. The c a r b o n chemical s h i f t s a s s i g n e d on t h e b a s i s of t h e a d d i t i v i t y p r i n c i p a l s and o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 3) (18).

9

a Fig. 5.

7

6

5

4

1 3

2

f

l3C-NMR Spectrum of Noscapine i n Deuterated Chloroform.

0

3

1

424

50

60

1

T

70

90

80

100 110 120 130 1 4 0 150 160

170

180

1

1

190 200 210 220 230 240 250 260 270 280 290 300

310

1

1 2

320 330

340 350 360 370 380 390 400 410 420 430 440 4 5 0 Fig. 7.

EI-Mass

Spectrum of Noscapine.

MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN

426

’

22

OCH3

Table 3. Carbon No.

c-1 c- 2

Carbon Chemical S h i f t s of Noscapine

Chemical S h i f t PPm 60.84 49.99 28.03 134.03 117.65 140.45 100.73 141.14 152.18 132.09 59.36

c-3

c-4 c-5 C-6

c-7 C-8 c- 9 c-10

c-11 2.5.4

(d) (t) (t) (s)

(d) (s) (t) (s)

(s) (s) (q)

Carbon No.

Chemical S h i f t Ppm

c-12 C-13 C-14 C-15 C-16 C-17 C-18 c-19 c-20 c-21 c-22

46.29 81.83 120.17 118.19 102.29 147.67 148.37 117.11 168.06 56.78 62.22

(q) (d) (s) (d)

(d) (s) (s) (s) (s) (4)

(q)

Mass Spectrum

The mass spectrum of n o s c a p i n e by e l e c t r o n impact i o n i z a t i o n and recorded on Ribermag R-10-10 mass equibbed w i t h d i r e c t i n l e t probe. ( F i g . 7) shows m o l e c u l a r i o n peak a b a s e peak a t m / e 220.

obtained which w a s spectrometer The spectrum and shows

The mass spectrum of n o s c a p i n e o b t a i n e d by butane chemical i o n i z a t i o n ( F i g . 8) shows a m o l e c u l a r i o n peak PI+ a t m / e 413 w i t h a r e l a t i v e i n t e n s i t y of 2.8% and a b a s e peak a t m / e 220. The most prominent f r a g m e n t s ,

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2t

1

290 300 31 0 320 330 340 350 360 370 380 390 4-00410 420 430 440 450 460 470 Fig.

8.

CI-Mass

Spectrum of Noscapine.

428

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN t h e i r r e l a t i v e i n t e n s i t i e s and s t r u c t u r e s a r e l i s t e d i n Table 4 . Other mass s p e c t r a l d a t a f o r p h t h a l i d e i s o q u i n o l i n e s was a l s o r e p o r t e d (19, 2 0 ) . Table 4 .

m/e 413

Mass Fragments of Moscapine Relative Intensity

96

Fragment

M+

2.8

221

220

195

8.0

OH

193 OCH3

429

NOSCAPINE

3.

P r eDara t i o n 3.1

I s o l a t i o n from Opium Noscapine o c c u r s up t o 11%n a t u r a l l y i n opium (Papaver sornniferum L . (Fam. papaveraceae) I t w a s f i r s t d i s c o v e r e d by Derosne i n 1803 ( 2 0 ) , and i s o l a t e d by Robinquet i n 1817 ( 2 1 ) . Noscapine can be s e p a r a t e d from o t h e r opium a l k a l o i d s by t h e procedure o u t l i n e d i n Scheme 4 (90).

.

Another p a t e n t method h a s been a l s o d e s c r i b e d f o r i t s i s o l a t i o n on a n i n d u s t r i a l scale ( 2 2 ) .

4.

S y n t h e s i s of Noscapine

4.1

By T i s s u e C u l t u r e Method Khanna e t a 1 (23) d e s c r i b e d a method f o r t h e s y n t h e s i s of noscapine a l o n g w i t h o t h e r a l k a l o i d s by t i s s u e c u l t u r e of Papaver somniferum Linn.

4.2

By Chemical Methods P e r k i n and Robinson (24) d i s c o v e r e d t h a t h e a t i n g a m i x t u r e of c o t a r n i n e 1 and meconine 1i n e t h a n o l r e s u l t e d i n a s m a l l y i e l d of noscapine 3 . The expected second isomer of noscapine-(because of t h e presence of 2 a s s y m e t r i c c e n t r e s ) w a s n o t found. The s y n t h e t i c noscapine was t h e n r e s o l v e d and t h e n o s c a p i n e o b t a i n e d shown t o b e i d e n t i c a l w i t h t h e n a t u r a l p r o d u c t . (Scheme 5).

A q u i t e e f f i c i e n t s y n t h e s i s of noscapine w a s developed by Hope and Robinson i n 1914 ( 2 5 ) , i n which c o t a r n i n e i s condensed w i t h iodomeconine 2 and t h e adduct w a s reduced w i t h sodium amalgum t o g i v e t h e d e s i r e d p r o d u c t , corresponding t o t h e n a t u r a l s e r i e s (Scheme 6 ) .

L

5.

B i o s y n t h e s i s of Noscapine I t has been p o s t u l a t e d t h a t t h e ph t h a l i d e i soqu i n o 1i n e s a r e formed i n n a t u r e by o x i d a t i v e m o d i f i c a t i o n of t e t r a h y d r o p r o t o b e r b e r i n e s , and p r e v i o u s work w i t h l a b e l e d precursors supports t h i s hypothesis (26).

430

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN Powdered Op i um

+

+

Shake w i t h warm calcium c h l o r i d e s o l u t i o n F i 1t e r Insoluble matter C (discard)

F i ltrate (hydroch l o r i des o f a 1ka l o i ds)

4

Reduce volume (evaporate under reduced p r e s s u r e ) t o syrupy 1 i q u i d

4

Add 10% NaOH s o l u t i o n Precipitates p k (noscapine, papaverine, thebaine)

4

a

l ine solution (morphine, codeine,

E x t rn aa c rt cw e i nt he ) ci h l o r o f o r m

Dissolve i n d i l u t e a l c o h o l

$.

Add a c e t i c a c i d t o make s l i g h t l y

0

C h 1 o r o f orm

Aqueous extract alkaline (containsolution i n g codeine) (morphine, narceine)

acidii

Add 3 volumes o f b o i l i n g water

A Solution

Precipitate (papave r ine, noscap ine)

4

(theba ine)

4

4

Further purification

Make a c i d i c

rz 1

\

Further pur i f ic a t i o n

Dissolve i n b o i l i n g 0.33%(aqueous)oxali c a c i d soln.

4

4

Make s l i g h t l y a l k a l i n e w i t h ammonia

A l l o w t o stand

Bring t o b o i l i n g A l l o w t o stand

C rys t a 1 s (papave r ine ac id oxalate)

4

Aqueous a c i d i c s o l n . ( s a l t s of morphine and na r c e i ne)

Sol u t i o n (noscap ine oxa 1 a t e )

+

Repeat

Precipitate (morphine)

I

Further p u r i f ic a t ion

Solution (narceine)

Make a l k a l i n e w i t h ammonia

Precipitate (noscapine)

+

1 i s o l u t on (discard)

+

Dissolve i n b o i l i n g alcohol Crystallization Scheme 4:

i

Further pur i f i cat ion

Isolation of

from powdered op i um .

NOSCAPINE

43 1

OCH

OCH3

2 Plecoriine

I

Cotarnine

D

C2H50H

0

0ch3 3

(2)-a-Narco t ine

Scheme 5

432

MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN

Cotarnine 1

Iodomeconine

\

2

\

CH 3

CH 3 CH 3O

Na/Hg H..

... *..s . 0

OCH3

OCIl

3

(+) -a-Narcotine

Scheme 6

433

NOSCAPINE

S e v e r a l f e e d i n g e x p e r i m e n t s ( 6 , 8 , 94 ) have been r u n t o e l u c i d a t e t h e b i o g e n e s i s of n o s c a p i n e i n Papaver somnif erum L . (Papaveraceae) When l a b e l e d (+) t y r o s i n e was f e d t o t h e p l a n t , r a d i o a c t i v e n a r c o t i n e l a b e l e d s p e c i f i c a l l y and e q u a l l y a t C-1 and C-3 was o b t a i n e d . The b e n z y l i s o q u i n o l i n e s y s t e m of n o s c a p i n e i s t h u s d e r i v e d b i o l o g i c a l l y from two Ar-C-C u n i t s which c a n a r i s e from t y r o s i n e .

.

The c a r b o n atoms t h a t a r i s e from t h e S-methyl of m e t h i o n i n e were c l e a r l y p i n p o i n t e d when, a f t e r f e e d i n g radioactive methionine, noscapine labeled a t t h e l a c t o n e c a r b o n y l , t h e m e t h y l e n e d i o x y g r o u p , and t h e N- and 0-methyl c a r b o n atoms w a s o b t a i n e d ( 2 7 , 2 8 ) . Progressing f u r t h e r along the biogenetic locus, t h e b e n z y l i s o q u i n o l i n e (+)- n o r l a u d a n o s o l i n e l a b e l e d C-1 l e d t o n o s c a p i n e a l s o l a b e l e d C-1 ( 2 9 ) . Even more s i g n i f i c a n t l y , when q u a d r u p l y l a b e l e d (+)- and (-) r e t i c u l i n e were f e d s e p a r a t e l y t o P. somniferum, i t w a s found t h a t b o t h enantiomers were i n c o r p o r a t e d i n t o n o s c a p i n e , b u t w i t h t h e (+)-isomer d o i n g so s l i g h t l y more e f f i c i e n t l y . E v i d e n t l y e p i m e r i z a t i o n of t h e wrong b e n z y l i s o q u i n o l i n e p r e c u r s o r must o c c u r , p r o b a b l y by o x i d a t i o n - r e d u c t i o n a t C-1. I n keeping w i t h t h i s c o n c l u s i o n c o n s i d e r a b l e l o s s of t r i t i u m o c c u r e d i n t h e c o u r s e of i n c o r p o r a t i o n of b o t h r e t i c u l i n e s . Another i m p o r t a n t o b s e r v a t i o n i s t h a t t h e l a c t o n e c a r b o n y l of t h e p h t h a l i d e i s o q u i n o l i n e must b e d e r i v e d from t h e N-methyl group of t h e b e n z y l i s o q u i n o l i n e p r e c u r s o r ( 2 7 , 29, 3 0 ) . F i n a l l y , i t h a s been found t h a t t h e f e e d i n g of l a b e l e d ( - ) - s c o u l e r i n e r e s u l t s i n t h e f o r m a t i o n of r a d i o a c t i v e noscapine. Protoberberines are, therefore, t h e precursors f o r t h e phthalideisoquinolines i n plants. S i g n i f i c a n t l y , ( - ) - s c o u l e r i n e , which p o s s e s s e s t h e same a b s o l u t e c o n f i g u r a t i o n a s ( + ) - r e t i c d i n e and ( - ) - a - n a r c o t i n e , w a s more t h a n one hundred t i m e s more e f f i c i e n t t h a n i t s enantiomer a s a p r e c u r s o r f o r ( - ) - a - n a r c o t i n e . The b i o g e n e t i c sequence i n p l a n t s i s , t h e r e f o r e , b e n z y l i s o qu i n o l i n e s +- t e t r a h y d r o p r o t o b e r b e r i n e s + p h t h a l i d e i s o q u i n o l i n e s . The b i o s y n t h e s i s of (-)-cC-narcotine i s shown i n Scheme 7 .

6.

Metabolism The m e t a b o l i s m of n o s c a p i n e was r e p o r t e d

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN

434

&:

Labeled t y r o s i n e

bCH3

*<:w Labeled noscapine

H $H3-S-(CH

1

Papaver

) -C-COOH 2 2 1 somn i f e r u m )

NH2 Labeled methionine

I

N\*

CH 3O

CH 3

- 0

OgH3 L a b e l e d nos cap i n e

Laheled ( a - n o r l a u d a n o s o l i n e Scheme 7

L a b e l e d n‘bscapine

435

NOSCAPINE

Scheme 7 ( c o n t i n u e d )

(4)

"H3

Labeled (+) - r e t i c u l i n e (Labeled (-) - r e t i c u l i n e somewhat less e f f i c i e n t )

R = H o r T Labeled n o s c a p i n e ( a p p r e c i a b l e l o s s of tritium)

CH30

HO

OCH3

Labeled (-) - s c o u l e r i n e

Labeled noscapine (Some t r i t i u m l o s s )

MOHAMMED A . AL-YAHYA AND MAHMOUD M . A . HASSAN

436

(31, 3 2 ) . Oral a d m i n i s t r a t i o n of to male r a b b i t s and e x a m i n a t i o n o f t h e 24 h o u r s u r i n e by p r e p a r a t i v e TLC and methane c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y r e v e a l e d t h e p r e s e n c e of two O-monodemet h y l a t e d compounds a s f r e e m e t a b o l i t e s 2 and 5, One 0 - d i d e m e t h y l a t e d d e r i v a t i v e 2 o r 5 and t h e i r c o n j u g a t e d forms. Noscapine g i v e n orally to rats was m e t a b o l i s e d t o di-0-demethyl-noscapine 3 or 4 , cotarnine 5 , h y d r o c o t a r n i n e 1,o x y c o t a r n i n e 2, a n d 0-demethylmeconine 5. T h e s e m e t a b o l i t e s were i s o l a t e d from u r i n e . A l l p o s s i b l e m e t a b o l i t e s of n o s c a p i n e a r e shown i n Scheme 8.

7.

Methods of A n a l y s i s

7.1

I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e d e s c r i b e d by t h e B r i t i s h Pharmacopoeia ( 1 9 8 0 ) . a)

The l i g h t a b s o r p t i o n , i n t h e r a n g e 230 t o 350 nm, of a 0.005 p e r c e n t w/v s o l u t i o n i n m e t h a n o l e x h i b i t s two maxima, a t 291 nm and 310 nm and a minimum a t 263 nm; r a t i o of t h e a b s o r b a n c e a t t h e maximum a t 310 nm t o t h a t a t t h e maximum a t 291 nm, a b o u t 1 . 2 .

b)

To 1 0 mg add 0 . 5 m l of s u l f u r i c a c i d and mix; a g r e e n i s h - y e l l o w s o l u t i o n i s formed which t u r n s r e d and f i n a l l y v i o l e t o n h e a t i n g .

c)

S o l u t i o n s i n organic s o l v e n t s , such as

methanol and c h l o r o f o r m , a r e l e v o r o t a t o r y ; aqueous a c i d i c s o l u t i o n s are d e x t r o r o t a t o r y . 7-2

M i c r o c r y s t a l Tests a)

According t o t h e method of C l a r k e and W i l l i a m s (33) , i n potassium chromate s o l u t i o n , n o scap i n e f o r m s f e a t h e r y r o s e t t e s o r b u n c h e s of b l a d e s , s e n s i t i v i t y b e i n g 1 i n 1500 ( F i g . 9 ) .

b)

I n sodium c a r b o n a t e s o l u t i o n r o s e t t e s and b u n c h e s of n e e d l e s a r e s e e n a t t h e same s e n s i t i v i t y (Fig. 10).

NOSCAPINE

437

Fig. 9 .

C r y s t a l s of Noscapine with Potassium Chromate S o l u t i o n .

Fig. 10.

C r y s t a l s of Noscapine with Sodium Carbonate S o l u t i o n .

MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN

43 8

Scheme 8 .

P o s s i b l e M e t a b o l i t e s of (-)-a-narcotine

J

be@ ‘ 0

0

OCH3

CH3

nos c a p i n e 1

5

0-demezhyla ted meconine

cotarnine

1

hydrocotarnine

CH3 c o t a r n i n e (pseudo b a s e form) OCH3

ox yc o t a r n i n e

0-d i d eme t h y l a t e d m e t a b o l i t e s

439

NOSCAPINE

7.3

T i t r i m e t r i c Methods The o f f i c i a l methods of d e t e r m i n i n g Noscapine a r e d e s c r i b e d by t h e B.P. (3) and U.S.P. ( 3 4 ) . 7.3.1

Non-Aaueous T i t r a t i o n The B.P. method :-

(3) d e s c r i b e s t h e f o l l o w i n g

D i s s o l v e 0.5 g i n 40 m l of anhydrous g l a c i a l a c e t i c acid previously neutralised t o c r y s t a l v i o l e t , warming g e n t l y . T i t r a t e w i t h 0.1 M p e r c h l o r i c a c i d u s i n g 0.25 m l of c r y s t a l v i o l e t s o l u t i o n a s i n d i c a t o r . Each m l of 0.1 M p e r c h l o r i c a c i d i s e q u i v a l e n t t o 0.04134 g of C22H23 NO,. The U.S.P. met hod :-

describes the following

D i s s o l v e about 1 . 5 g of Noscapine, a c c u r a t e l y weighed, i n 25 m l of g l a c i a l a c e t i c a c i d . Add 25 m l of d i o x a n e and 5 d r o p s of c r y s t a l v i o l e t T . S . , and t i t r a t e with 0.1 N perchloric acid i n g l a c i a l a c e t i c a c i d t o t h e end-point change from p u r p l e t o b l u e . Perform a b l a n k d e t e r m i n a t i o n , and make any n e c e s s a r y c o r r e c t i o n . Each m l of 0.1 N perchloric acid is equivalent t o 41.34 mg of C22H23N07. Another method w a s d e s c r i b e d by T u t h i l l e t a l . (35) u s i n g m a l a c h i t e g r e e n a s b e t t e r indicator than c r y s t a l v i o l e t . 7.3.2

Polarographic T i t r a t i o n a)

Using a dropping mercury e l e c t r o d e a s i n d i c a t o r n o s c a p i n e h y d r o c h l o r i d e c a n be t i t r a t e d w i t h Cadmium I o d i d e i n s o l u t i o n of n e u t r a l s a l t s ( 0 . 1 t I KNO N a C l or 3’ Na2S04) ( 3 6 ) .

b)

Dusinsky (37) d e s c r i b e d a method where noscapine c a n be t i t r a t e d i n a l k a l i n e

440

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN s o l u t i o n (1.25 N NaOH) showing depression i n t h e polarographic curve a t 1.5V. Sodium a l i z a r i n s u l p h o n a t e h a s been used f o r t h e d e t e r m i n a t i o n of n o s c a p i n e (38). A p o t e n t i a l of - 0 . 6 5 V was used and t h e t i t r a t i o n medium was 0.3 N K C 1 a d j u s t e d t o a pH of 4 - 6 . SoucKova and S;ka (39) s t a t e d a method using t u n g s t o s i l i c i c acid. This acid w a s found e s p e c i a l l y u s e f u l s i n c e t h e r e a c t i o n i s v e r y s e n s i t i v e and prec i p i t a t i o n i s immediate. The p o l a r o graphy a l s o gave t h e c o m p o s i t i o n of t h e t u n g s t o s i l i c i c acid organic base complex. The poor s e l e c t i v i t y of T u n g s t o s i l i c i c a c i d i s due t o i t s h i g h s e n s i t i v i t y which a l l o w s a c c u r a t e d e t e r m i n a t i o n of 10-20 mg of b a s e . A 0.01 M aqueous s o l u t i o n of t u n g s t o s i l i c i c a c i d i s used w i t h a dropping mercury c a t h o d e and S . C . E . anode a t 0 . 6 5 V . The pH of s o l u t i o n a d j u s t e d w i t h HC1 ( 0 . 1 t o 0 . 6 N ) . P l o t of c u r r e n t vs a c i d used c o n s i s t s of two s t r a i g h t l i n e s and t h e i n t e r s e c t c o n s i d e r e d as t h e e q u i v a l e n c e p o i n t . Another method f o r p o l a r o g r a p h i c t i t r a t i o n was a l s o r e p o r t e d ( 4 0 ) . T h i s method is based on t h e f o r m a t i o n of complex mercury compounds. S o l u t i o n of K2HgI4 c o n t a i n i n g a n e x c e s s of i o d i d e i s t h e most s u i t a b l e f o r t h e determinat i o n . The t i t r a t i o n i s c a r r i e d o u t w i t h a d r o p p i n g mercury e l e c t r o d e a t t h e p o t e n t i a l -0.8 V t o - 0.9 V (VS. t h e S . C . E . ) w i t h 0 . 1 PI KN03 o r 0 . 1 M H SO a s s u p p o r t i n g e l e c t r o l y t e . 2 4

7.3.3

P o t e n t iometr i c T i t r a t i o n Tungsten rod w a s used a s i n d i c a t o r e l e c t r o d e i n t h e p o t e n t iometr i c t i t r a t i o n of n o s c a p i n e i n a 1 : 6 m i x t u r e of a c e t i c a c i d : a c e t i c anhydride ( 4 1 ) .

441

NOSCAPINE

7.4

Complexometric Noscapine is p r e c i p i t a t e d from 0 . 5 N H C 1 w i t h 0.028 M Bi-EDTA and 0.112 M K I forming iodobismuthate complexes and EDTA i s being set f r e e (42). After c e n t r i f u g a t i o n t h e f r e e EDTA i s determined i n a n a l i q u o t of t h e supern a t e n t l i q u i d w i t h 0.01 M ZnSO4 i n pH 9 . 1 b o r a t e b u f f e r and Eriochrome b l a c k T as i n d i c a t o r . T e r t i a r y amines, q u a t e r n a r y ammonium s a l t s o r analogous sulphonium, phosphonium and arsonium compounds i n t e r f e r e i n the determination.

7.5

Spectrophotometric 7.5.1

Colorimetric

a ) Yoichi and Sano (43) d e s c r i b e a method f o r a n a l y s i s of noscapine i n mixed pharmaceutical preparations. The sample is mixed w i t h a s o l u t i o n of chromotropic a c i d 0.2% i n 70% (v/v) H3PO4 a c i d and h e a t e d a t 100°C f o r 30 m i n u t e s and t h e e x t i n c t i o n i s measured a t 570 nm. A c a l i b r a t i o n c u r v e i s r e c t i l i n e a r f o r 30 t o 150 pg of noscapine h y d r o c h l o r i d e per m l . b) Solochrome Green V 150 ( C . I . Mordant Green 15) h a s been used a s aqueous 1 mM s o l u t i o n . The complex formed by noscapine is e x t r a c t e d i n t o chloroform and t h e absorbance i s measured a t 520 nm ( 4 4 ) . c ) Another method f o r t h e q u a n t i t a t i v e s e p a r a t i o n of p a p a v e r i n e from n o s c a p i n e i n m i x t u r e s w a s a l s o r e p o r t e d (45). T h i s method i s based on t h e f o r m a t i o n of a n insoluble papaverine r e i n e c k a t e i n acid s o l u t i o n i n t h e p r e s e n c e of e x c e s s chloroform. Procedure T r i t u r a t e t h e sample (4.5 g) w i t h g l a c i a l a c e t i c a c i d (25 ml) followed by H 2 0 ( 20 ml) and f i l t e r . E x t r a c t a 1 0 m l a l i q u o t w i t h

442

MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN

CHC13 (8 X 1 0 ml) and wash e a c h e x t r a c t i n t u r n w i t h H20 (15 m l ) , H20 (15 ml) p l u s NaOH soln. ( 1 : 1) containing a l i t t l e NaHS03 (15 m l ) , H 2 0 ( 1 5 m l ) , 0 . 1 M H2SO4 (15 m l and 1 0 ml) and 0.05% N a H C 0 3 s o l n . (10 m l ) . E v a p o r a t e t h e combined washed e x t r a c t s t o d r y n e s s o n a water b a t h . D i s s o l v e t h e r e s i d u e i n CCl4 (50 ml) , s t r a i n through cotton-wool and p a s s t h r o u g h a column of Ca(OH)2. Wash t h e column w i t h C C l 4 (2 X 1 0 ml) and e x t r a c t t h e combined CCl4 f r a c t i o n s w i t h 0 . 1 N H C 1 ( 2 X 1 0 m l ) . Shake t h e H C 1 s o l n . w i t h CHC13 (10 ml) f o r 10 m i n . , add 2% ammonium r e i n e c k a t e s o l n . ( 1 0 m l ) , shake f o r 3 0 min. and f i l t e r through s i n t e r e d g l a s s . To d e t e r m i n e papaverine d i s s o l v e t h e ppt. i n acetone and measure t h e e x t i n c t i o n a t 525 mu. To d e t e r m i n e n o s c a p i n e , shake t h e CHC13 l a y e r of t h e f i l t r a t e w i t h 0.25% AgN03soln. ( 4 0 m l ) , s e p a r a t e and f u r t h e r e x t r a c t w i t h CHC13 (2 X 1 0 m l ) ; s t r a i n t h e combined CHC13 f r a c t i o n s through cotton-wool, d i l u t e t o 250 m l , and e i t h e r measure t h e e x t i n c t i o n a t 310 mp o r e v a p o r a t e and t i t r a t e w i t h 0.05 N H C l O 4 i n g l a c i a l a c e t i c a c i d . d) Thomas d e s c r i b e d a method f o r d e t e r m i n a t i o n of some d r u g s c o n t a i n i n g a t e r t i a r y - a m i n e group ( 4 6 ) . The d r u g i s h e a t e d w i t h 10% malonic a c i d i n a c e t i c a n h y d r i d e a t 800 f o r 1 5 min. and, a f t e r d i l u t i o n w i t h e t h a n o l , t h e e x t i n c t i o n i s measured a t 333 nm. t h e l i m i t of d e t e c t i o n f o r n o s c a p i n e hydroDosage c h l o r i d e was 1 0 t o 30 ng m l - 1 . forms r e q u i r e p r e l i m i n a r y e x t r a c t i o n of t h e drug.

7 . 5.2

Infra-red

et a1 (47) d e s c r i b e d a n i n f r a - r e d Bakre s p e c t r o s c o p i c method f o r t h e d e t e r m i n a t i o n of t h e o r i g i n of opium as w e l l a s a s i m u l t a n e o u s a s s a y of n o s c a p i n e , t h e b a i n e and papav e r i n e . 4.5 g f i n e l y ground sample w a s t i t u r a t e d f o r 20 min. i n 25 m l water w a s s l o w l y added w i t h c o n t i n u o u s s t i r r i n g and t h e

443

NOSCAPINE

resulting solution was filtered. 10 m l A l i q u o t of f i l t r a t e w a s e x t r a c t e d f o u r t i m e s i n t o 1 0 m l c h l o r o f o r m and e a c h e x t r a c t w a s washed w i t h 1 0 m l water, 25 m l of 0.2% sodium b i s u l p h i t e i n 30% aqueous sodium h y d r o x i d e , 1 0 m l water and a g a i n 1 0 m l water. The combined c h l o r o f o r m s o l u t i o n was f i l t e r e d t h r o u g h c o t t o n wool and e v a p o r a t e d . The r e s i d u e i s d r i e d i n a d e s i c a t o r t h e n mixed w i t h anhydrous c a r b o n t e t r a c h l o r i d e , f i l t e r e d through s i n t e r e d g l a s s and d i l u t e d t o 25 m l . The I R i s examined from 1100 cm-1 t o 1900 c m - l i n a 1 mm sodium c h l o r i d e c e l l , noscapine b e i n g measured a t 1767 cm-l. The a b s o r b a n c e i s compared w i t h a b s o r b a n c e s of s o l u t i o n s of known c o n c e n t r a t i o n s . Other I R methods f o r d e t e r m i n a t i o n of i s o q u i n o l i n e a l k a l o i d s were a l s o r e p o r t e d (48, 4 9 ) . 7.5.3

Ultra-Violet T e t r a p o n a m i x t u r e of t h e h y d r o c h l o r i d e s of Morphine, Noscapine, c o d e i n e and p a p a v e r i n e w a s a n a l y s e d by J e n s e n ( 5 0 ) . Morphine w a s s e p a r a t e d by e x t r a c t i o n w i t h c h l o r o f o r m from s t r o n g a l k a l i n e s o l u t i o n and d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h NaN02 a t 440 run. The o t h e r a l k a l o i d s were s e p a r a t e d by T L C on K i e s e l g e l CF 254 w i t h e t h a n o l : benzene 1 : 4 a s s o l v e n t . The s p o t s ( l o c a t e d i n U.V. r a d i a t i o n ) were e x t r a c t e d w i t h methanol and determined a t 215 run f o r c o d e i n e , a t 279 nm f o r p a p a v e r i n e and 312 nm f o r n o s c a p i n e .

7.5.4

Atomic A b s o r p t i o n An i n d i r e c t method f o r t h e a n a l y s i s of Noscapine i n d r u g s w a s r e p o r t e d ( 5 1 ) . A complex i s formed between Noscapine and Reinecke s a l t i n t h e p r e s e n c e of t a r t a r i c a c i d a t pH 1 . 7 . This is extracted i n t o c h l o r o f o r m and Noscapine i s d e t e r m i n e d i n d i r e c t l y by measuring chromium c a t i o n by a t o m i c a b s o r p t i o n s p e c t r op ho tome t er y

.

444

MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN 7.5.5

Spectrofluorimetdc a ) Noscapine h a s been determined i n m i x t u r e s of opium a l k a l o i d s (52) by measuring t h e f l u o r e s c e n c e a t 375 run ( e x c i t a t i o n a t 315 nm). The sample i s b u f f e r e d a t pH 9 i n 0 . 1 N s u l p h u r i c a c i d and 0 . 1 N sodium hydroxide. Noscapine i s e x t r a c t e d i n chloroform and a p o r t i o n of t h i s e x t r a c t i s t r e a t e d w i t h t r i c h l o r o a c e t i c a c i d i n chloroform t o quench t h e f l u o r e s c e n c e of papaverine. A s t a n d a r d s o l u t i o n of 2-aminopyridine i n 0 . 1 N s u l p h u r i c a c i d is a l s o measured a t 375 nm ( e x c i t a t i o n a t 315 nm). For t h e c a l c u l a t i o n each f l o u r e s c e n c e r e a d i n g on t h e t e s t s o l u t i o n i s c a l c u l a t e d a s a p e r c e n t a g e of t h a t f o r t h e s t a n d a r d and r e f e r r e d t o c a l i b r a t i o n g r a p h s prepared s i m i l a r l y f o r Noscapine. Sub-microgram amounts of Noscapine c a n b e determined without preliminary separation. b) Vedso s t a t e d a method (53) f o r t h e d e t e r m i n a t i o n of Noscapine i n plasma and urine. I t involves e x t r a c t i o n of 1 m l sample a t pH 1 0 i n t o e t h y l e t h e r and r e - e x t r a c t i o n w i t h d i l u t e HC1. The a c i d i s n e u t r a l i s e d and t h e s o l u t i o n was a d j u s t e d t o PH 9.2 w i t h a borax b u f f e r . Fluorescence i s measured through 480 t o 580 mu f i l t e r ( e x c i t a t i o n a t 365 mu) b e f o r e and a f t e r a u t o c l a v i n g a t 120OC f o r 3 0 min. S t a n d a r d s ( 0 t o 2.5 pg cm-3) are a l s o measured i n t h e same way. I t was s t a t e d t h a t c o n c e n t r a t i o n s from 0.05 ug p e r m l can be determined and a l t h o u g h t h e p r e s e n c e of morphine gave an i n c r e a s e i n f l o u r e s c e n c e about h a l f t h a t f o r n o s c a p h e , c o d e i n e , n a r c e i n e and papaver i n e d i d n o t i n t e r f e r e

.

c ) A r e a c t i o n m i x t u r e of 10%malonic a c i d i n

a c e t i c a n h y d r i d e was used i n a method r e p o r t e d by Rao and Tandon ( 5 4 ) . I n t e r f e r e n c e was caused by T e r t i a r y amines, glucose,magnesium a c e t a t e and some i n o r g a n i c s a l t s , b u t n o t by d i e t h y l a m i n e , a n i l i n e , benzoic a c i d , a s p i r i n and s a c c h a r i n .

NOSCAPINE

445

7.5.6

Nuclear Magnetic Resonance A known amount of t - b u t y l a l c o h o l w a s added as a s t a n d a r d t o n o s c a p i n e i n e t h a n o l f r e e c h l o r o f o r m and t h e peaks a t 3 . 8 3 , 4.00 and 4 . 0 5 ppm; c o r r e s p o n d i n g t o t h e n i n e methoxy group p r o t o n s of n o s c a p i n e were i n t e g r a t e d a l o n g w i t h t h e peak a t 1 . 3 ppm c o r r e s p o n d i n g t o t h e n i n e methyl-group p r o t o n s of t - b u t y l a l c o h o l ( F i g . 11). The amount of n o s c a p i n e i s c a l c u l a t e d from t h e i n t e g r a t i o n r a t i o and t h e known amount of s t a n d a r d ( 5 5 ) .

7.5.7

Mass Noscapine w a s i d e n t i f i e d i n opium (92) w i t h o u t any p r i o r s e p a r a t i o n . Samples a r e introduced d i r e c t l y i n t o t h e ion s our c e us ing a s o l i d sampling probe. Reagent g a s e s were i s o b u t a n e and w a t e r , mass s p e c t r a l measurement was a t m / e 220. Arnold (93) d e s c r i b e d a G C /M S method f o r t h e d e t e r m i n a t i o n of n o s c a p i n e i n opium preparations.

7.6.

Chromatographic

7.6.1

Paper Chromatography The paper-chromatographic method f o r t h e d e t e c t i o n of a l k a l o i d s , e . g . , c o d e i n e , v e r a t r i n e , q u i n i n e and n o s c a p i n e i n s e v e r a l f o o d s ( a 100 g sample) i s d e s c r i b e d . For t h e p r e l i m i n a r y e x t r a c t i o n of t h e a l k a l o i d s , add t o t h e sample 300 m l of e t h a n o l a c i d i f i e d t o l i t m u s p a p e r w i t h H C 1 . D i g e s t o n a water b a t h a t 400 f o r 24 h r . , f i l t e r and r e t a i n t h e f i l t r a t e . Add 100 m l of acidified ethanol to the residue, d i g e s t f o r a n o t h e r 1 2 h r . and f i l t e r a g a i n . Combine t h e f i l t r a t e s and remove t h e a l c o h o l by e v a p o r a t i n g on a w a t e r b a t h a t 4 0 0 . Add 30 m l of water and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 5 0 m l ) . Add NaOH s o l n . t o t h e a q . l a y e r till i t is a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 50 ml). Evaporate t h e e t h e r e x t r a c t t o 50 m l , t r a n s f e r i t t o a s e p a r a t i n g - f u n n e l

446

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN and wash w i t h 2N HCl(3 X 20 m l ) . To t h e combined a c i d washings add NaOH till t h e y a r e a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l e t h e r ( 3 X 50 m l ) . E v a p o r a t e t h e e t h e r c o m p l e t e l y and d i s s o l v e t h e extract i n 5 m l of e t h a n o l . R e t a i n t h i s a l c o h o l i c s o h . f o r chromatography by t h e d e s c e n d i n g t e c h n i q u e on Whatman No. 1 p a p e r . Apply d r o p s of t h e t e s t and s t a n d a r d s o l n . t o t h e p a p e r and impregnate i t s whole area above t h e s t a r t i n g l i n e w i t h a f r e s h l y p r e p a r e d e t h a n o l i c s o h . of formamide (1 : 1). Dry t h e paper between two f i l t e r p a p e r s , t h e n a t 400 f o r 3 0 min. Spray t h e s t a r t i n g p o i n t s w i t h t h e e t h a n o l i c s o l n . of formamide (1 : 1) and a f t e r 10 min. t r a n s f e r t h e paper t o a g l a s s c o n t a i n e r c o n t a i n i n g CHC13 a t 250. The development i s complete i n 2.5 h r . Dry t h e chromatogram a t 1050 and s p r a y w i t h a s o h . of potassium i o d o p l a t i n a t e , washing o f f t h e excess w i t h water i n o r d e r t o o b s e r v e t h e s p o t s on t h e w h i t e background. The f o l l o w i n g amounts of a l k a l o i d s can b e d e t e c t e d - c o d e i n e 0.01 t o 0.3 mg, q u i n i n e 0.005 t o 0.015 mg, v e r a t r i n e 0.1 t o 0.15 mg and noscapine 0.05 t o 0.075 mg. ( 5 6 ) . T a b l e 5 d e s c r i b e s methods used i n noscapine a n a l y s i s . A n a l y s i s of noscapine i n opium by paper chromatography and s p e c t r o p h o t o m e t r y involved e x t r a c t i o n of noscapine and measurement of t h e e x t i n c t i o n of t h e s p o t a t 290 mu and comparison w i t h s t a n d a r d s ( 5 7 ) . Other method was a l s o d e s c r i b e d ( 5 8 ) . 7.6.2

Thin Layer Chromatography T h i s t e c h n i q u e h a s been used e x t e n s i v e l y f o r a n a l y s i s of Opium and i t s p r e p a r a t i o n s (63 - 7 1 ) . T a b l e 6 - g i v e s a resume of t e c h n i q u e s and T a b l e 7 - shows s p r a y r e a g e n t s and methods used. S t a h l and co-workers have proposed a s t a n d a r d p r o c e d u r e f o r t h e s e p a r a t i o n of opium a l k a l o i d s ( 6 3 ) . Dried opium ( 0 . 1 g)

... I

S O

. . . .

. - . .

. . . . I . . . . ~ " . . ~ . . . . ~ . . " . ~' I" ' I

I

'

400

300

zbo

I

I

I o ni

.

I

100

H3

1

.

1

I

l . . l . . . ' l . . . ' l . . . . i . . - . l

.

1 . .

.

1

. I . .

1 .

. I

I . .

..

I

II

T a b l e 5.

1 S t a t i o n a r y Phase Paper

Paper

2 Technique Two d i m e n s i o n a l

Two d i m e n s i o n a l

Paper Chromatography Used € o r Noscapine. 3

4

Mobile P h a s e

1. Water s a t . b u t a n o l - a c e t i c acid 5:l 2. e t h e r - 0 . 1 M a c e t i c a c i c 5: 2

6

5 Comment Alkaloids i n Tetrapon 11

1. Dioxan-Formic a c i d - w a t e r 90:0.5 : 9 . 5 2. n - B u t a n o l - a c e t i c a c i d 5:l

11

11

Paper

One d i m e n s i o n a l

1. Dioxan-Formic 90:0.5:9.5

Paper S & S 204 3b.

Ascending o r descending

25% (NH4)2 SO4 i n 0 . 5 N H C 1

Whatman No. 1 paper b u f f e r e d a t pH 3.5

D e s c end i n g

Isobutylalcohol - toluene s a t u r a t e d w i t h water 1 : 1.

Paper

One d i m e n s i o n a l

Butyl acetate - acetic acid 47:9:28:16

acid-water

- butanol - water

11

Separation of n o s c a p i n e from papaver i n e enhancec b e c a u s e of u s e of b u t y l acetate.

rferenct

T a b l e 5. c

I 1

\D

I

2

(contd

... .)

3

Paper

One d i m e n s i o n a l

Upper l a y e r of a m i x t u r e n-butanol-acet i c a c id-water 5:1:4

Wha tman N o . 1 impregnated above s t a r t i n g l i n e with f ormamidee t h a n o l 1:l then d r i e d between f i l t e r papers t h e n a t 40°C f o r 30 min.

D esc end

Chloroform

4

5

Starting points w e r e sprayed w i t h f ormamide i n e t h a n o l 1:l a f t e r drying t h e paper

6

(56)

T a b l e 6.

S t a t i o n a r y Phase K i e s e l g e l HF

254

TLC Techniques Used f o r Noscapine

Technique Normal chamber

Mobile P h a s e

Rf

Tolune-acetone 95% e t h a n o l 25% aq. NH3

20 : 20 : 3 : 1

P

-

S i l i c a g e l G-Na2C03

tI

Chloroform 4 : l

Silica gel G.

II

E t h y l acetate

K i e s e l g e l 60

It

Chloroform Benzene Acetone 3 : 3 : 1

Silicagel G

I1

Benz ene-methanol

Silica gel G

I1

B u t a n o l - A c e t i c acid-H 0 2 3 : l : l

S i l i c a g e l G impreg. 4% Na2C03

11

Chloroform 4 : l

Silica gel

11

Chloroform-isopropyl a l c o h o l 10% aq.NH3 30 : 10 : 1 Benzene-methanol 4 : 1

Ethanol

VI

0

-

4 : l

Silica gel G

-

Ethanol

Table 7.

TLC S p r a y R e a g e n t s and Methods Used € o r D e t e c t i o n of Noscapine

Reagent

Procedure ~

5% 3,5-dichloro-p-benzoquinonechlorimine i n i s o p r o p y l alcohol

~~

A f t e r s p r a y i n g s p r a y w i t h aqueous

NH3 1 : 1 and o b s e r v e i n d a y l i g h t and u l t r a v i o l e t .

4% Hg(N03)2 i n 3% HN03

After spraying t h e p l a t e i s heated 15 min. a t l l O O C and o b s e r v e d i n d a y l i g h t . D e t e c t i o n l i m i t 2 vg

1. 3% H202 s o l u t i o n 2. 5% K4Fe(CN)6 s o l u t i o n

P l a t e i s f i r s t d r i e d 1 0 min. a t 100°C t h e n s p r a y e d w i t h 1. t h e n d r i e d 1 0 min. a t 100°C and s p r a y e d w i t h 2 and d r i e d 1 0 min. a t 100°C. Brown s p o t s i n t e n s i f i e d t o r e d . D e t e c t i o n l i m i t 1 0 pg.

2.6 g C@(N@3)2 d i s s o l v e d i n 2 m l anhydrous a c e t i c a c i d i s added t o 4 . 4 g o f NaN02 d i s s o l v e d i n 1 0 m l H20 t h e n 20 ml a c e t i c a c i d and 50 m l H 0 i s added t o m i x t u r e . 2

After spraying, t h e p l a t e is heated a t 105 OC f o r 10 min. S p o t s a r e s t a b l e f o r several h o u r s . Noscapine a p p e a r s a s b l u e - g r e e n f l o u r e s c e n t s p o t when viewed under u l t r a - v i o l e t l i g h t .

4% c i t r i c a c i d i n

P l a t e i s h e a t e d a t 80 O C f o r 1 0 min. and viewed i n d a v l i g h t ( d e t e c t i o n l i m i t 5 pg) and i n u l t r a - v i o l e t r a d i a t i o n ( d e t e c t i o n l i m i t 0.5 Up).

a c e t i c anhydride

Ref.

452

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN w a s powdered and t r i t u r a t e d w i t h 5 m l of 70% e t h a n o l . The m i x t u r e w a s warmed a t 50 - 600 f o r 30 min. t h e n f i l t e r e d and d i l u t e d t o 1 0 m l w i t h 70% e t h a n o l . T i n c t u r e of opium (1 m l ) was d i l u t e d w i t h 9 m l 35% e t h a n o l . Three p o r t i o n s ( 5 , 1 0 and 20 u l ) of opium s o l u t i o n and similar p o r t i o n s of s t a n d a r d s o l u t i o n were a p p l i e d t o a l a y e r of K i e s e l g e l H F254 and chromatograph was developed t o 1 5 c m w i t h t o l u e n e - a c e t o n e - 95% e t h a n o l 25% a q . NH3 (20 : 20 : 3 : 1 ) . For d e t e c t i o n , t h e p l a t e w a s h e a t e d a t 110% f o r 1 0 min. and t h e s e p a r a t e d zones l o c a t e d i n u l t r a v i o l e t r a d i a t i o n . The s p o t s a r e t h e n sprayed u s i n g a modified D r a g e n d o r f f ' s r e a g e n t and t h e n w i t h 0.05 N t o 0 . 1 N s u l p h u r i c a c i d . A l s o polyamide h a s been used a s l a y e r f o r chromatography ( 7 6 ) . Where poly-E-caprolactam r e s i n (Amilan CM 10075) was used. Development f o r 2 h r . i n cyclohexane-ethyl a c e t a t e p r o p y l a l c o h o l - Me2NH 3 0 : 2.5 : 0.9 : 0 . 1 showed n o s c a p i n e a t Rf 0 . 6 1 and development i n H20-ethanol - Me2NH 88 : 1 2 : 0 . 1 n o s c a p i n e had Rf 0.00.

7.6.3

Gas Liquid Chromatography

D e r i v i t i z a t i o n of samples i n c l u d e d a c e t y l a t i o n using acetic anhydride i n p y r i d i n e (77) and t r e a t i n g sample w i t h t r i m e t h y l s i l y l acetamide and t r i m e t h y l c h l o r o e t h a n e ( 6 8 ) . I n t e r n a l s t a n d a r d s used were h i s t a p y r r o d i n e h y d r o c h l o r i d e , o e s t r a d i o l v a l e r a t e (78) Phenazone (79) and S q u a l i n e ( 7 7 ) . Another method was a l s o r e p o r t e d ( 8 0 ) . Column t y p e s e t c . a r e r e p o r t e d i n T a b l e 8. 7.6.4

HiPh Performance Liquid Chromatography S e p a r a t i o n s of p h a r m a c e u t i c a l s combined i n v a r i o u s f o r m u l a t i o n s by HPLC on S e p h e r o s i l 5 Um i s o c r a t i c a l y h a s been r e p o r t e d ( 8 1 ) . Noscapine i n a n t i - c o l d p r e p a r a t i o n s w a s s e p a r a t e d on a column of H i t a c h i g e l 3011-0(82)

T a b l e 8.

Column Type

S t a t i o n a r y Phase

4 f t x 4 m m glass

D i a p o r t S (80-100) mesh

1 . 5 m x 2.3 mm g l a s s

Chromosorb G-HP AW-DMCS (80-100) mesh

GLC ConditionsUsed f o r Noscapine

3 0 cm

Gas-Chrom 0 (100-120 mesh)

3

min

-1

24OoC

SE-30

40 cm

3

min

-1

HI-EFF 8 BP

-

Supelcoport (80-100 mesh) 4 f t x 3mm od g l a s s

Temperature

Flow-ra t e

2% OV 101

N2

30 cm

3

min

-1

tg

I e t e ct o r

Flame i o n i z at i o r

15OoC 235OC a t 1 . 2 5 C min-1

11

2 2 5OC-2 7 O°C a t 2 0 min-1 ~

11

1 8 O o C 5 min. then 7 ' ~ min-l t o 25OoC

11

Ref,

454

MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN

u s i n g methanol : 28% aqueous NH3 9 9 : l a s e l u t i n g s o l v e n t and d e t e r m i n a t i o n by spectrophotometry a t 230 nm o r 250 nm. Paracetamol, p h e n a c e t i n , d i p y r o n e , a s p i r i n , c a f f e i n e , etenzamide and m e t h y l e p h e d r i n e d i d n o t i n t e r f e r e . Other methods were a l s o d i s c r i b e d (83, 8 4 ) . 7.6.5

Ion-Exchange Chromatography Knox and Jurand r e p o r t e d a method (85) f o r t h e s e p a r a t i o n of n o s c a p i n e on d r y packed column of Zipax SCX o r SAX (37-44 um) b o r a t e b u f f e r s a t pH 9.2-9.8 c o n t a i n i n g 4% a c e t o n i t r i l e and 1%propanol were used a t 500 t o 1500 l b p e r s q . i n . t o e l u t e n o s c a p i n e .

7.6.6

Ligand-Exchange Chromatography P o r a g e l P.T. r e s i n was used t o s e p a r a t e a l k a l o i d s and n o s c a p i n e h a s been a n a l y s e d on t h i s r e s i n . 0.06 M-aqueous NH3 i n 33% e t h a n o l w a s used as e l u a n t and t h e e l u t i o n volume f o r n o s c a p i n e w a s 6.8 times t h e bulk column volume ( 8 6 ) .

7.6.7

P a r t i t i o n Chromatography I n t h e a s s a y of T e t r a p o n by p a r t i t i o n chromatography t h e s t a t i o n a r y phase i s a phosphate b u f f e r ( 8 7 ) . 5 m l of 0 . 2 N NaOH i s added t o 0.4 g of papaveretum i n 20 m l H 2 0 . The m i x t u r e i s e x t r a c t e d twice w i t h a m i x t u r e of 1 0 m l of c h l o r o f o r m i n 3 0 m l e t h e r and t h e n w i t h 1 0 m l of chloroform. The f i l t e r e d e x t r a c t s a r e e v a p o r a t e d t o 0.5 1 . 0 m l and d i l u t e d w i t h 25 m l of e t h e r b e f o r e t r a n s f e r t o t h e p r e p a r e d column, 200 m l water s a t u r a t e d e t h e r is used t o e l u t e n o s c a p i n e . Other a s s a y w a s a l s o r e p o r t e d ( 8 8 ) .

7.6.8

Paper E l e c t r o p h o r e s i s Due t o f o r m a t i o n of m o l e c u l a r complexes between n o s c a p i n e and 7-(2-hydroxyethyl) t h e o p h y l l i n e , t e t r a m e t h y l u r i c a c i d and 7-carboxymethyltheophyline, n o s c a p i n e h a s

455

NOSCAPINE

been separated from other isoquinoline alkaloids by reversed - phase paper chromatography and electrophoresis. Britton - Robinson buffer pH 3.5 to 4 was used as the mobile phase and o-xylene as stationary phase ( 8 9 ) . Another method was also reported (1).

ACKNOWLEDGEMENTS The authors would like to thank the technicians, Robert Hutchison, K.N. Ludhi and the Research assistant Syed Rafatullah o f the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia for their kind technical assistance for the preparation of the manuscript.

456

8.

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.

1.

235,

PENICILLIN-G BENUTHINE Franz Kreuzig

1. Description 1. I Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 1.3 Compendia1 References 1.4 Definition of International Unit 1.5 Content 2. Production 3. Physical Properties 3.1 Solubility 3.2 Melting Range 3.3 Optical Rotation 3.4 Crystal Properties 3.5 Ultraviolet Spectrum 3.6 Infrared Spectrum 3.7 Nuclear Magnetic Resonance Spectrum 3.8 Mass Spectrum 4. Stability, Degradation, Artefacts 5. Biopharmaceutics 5.1 Pharmacokinetics 5.2 Metabolism 6. Methods of Analysis 6.1 Identification 6.2 Iodometric Titration 6.3 Nonaqueous Titration 6.4 Spectrophotometric Assay 6.5 Microbiological Assay 6.6 Thin-Layer Chromatography 6.7 High-Performance Liquid Chromatography 7. Acknowledgements 8. References

Analytical Profiles of Drug Substances Volume I I

463

464 464 464 465 465 465 465

466 466 467 467 467 467 469 469 469 472 472 472 474 474 474 475 476 476 477 478 479 480 480

Copyrighl 0 1982 by 'The American Pharmaceutical Association ISBN 0-12-260811-9

464

FRANZ KREUZIG

i.

DESCRIPTION

- I. I.

Name, Formuia, Moiecuiar Weight Chemi c a i Names -------------

-7

4-Thia-i -azabi c y c i o 1-3.2.0. 3,3-dimethyi-7-oxo-6 5a,6B)-7-,

heptane-2-carboxyi ic a c i d ,

[-(phenyiacetyi)amino - 7 - /-- 2

S-(2a,

compd. w i t h N,N'-bis(pheny methyi ) - i Y 2 - e t h a n e -

diami ne ( 2 : i ) , t e t r a h y d r a t e .

Generic-names Benzathi ne Peni c i iiin G, D i benzyi e t h y enedi ami ne-Di peni c i i i i n G, Benzathine B e n z y i p e n i c i i i i n , P e n i c i i i i n G Benz a t h i ne. Trade names -----------

, C i t o c y i iina , Diami n o c i i i i n a , Wyci iii n a , Beaci i i in , Pen-

Tardociiiin, Extenciiiine

P a n s t r i i i i n e , Penidurai

duran, B i c i i i i n , Moidamin, Retarpen, Pronapen, T r i - P e n i i e n t e , Debeci i7 i n , Retaci iii n , Penadur.

S ~ c H ~ - c o - N H - C H- CH/ 4\

'

4

C-N-

' 7

C48H56N608S.4 H20

P

3

CH2

'f'CH3 'CHCOO

2

+I

1

CH2 2

Moiecuiar Weight: 9 8 i , i 9

CA-Registry no. 1-1538-09-6-7

i. 2 .

I+

HN-CH2-CH2-NH.4 H20

Appearance, Color, Odor White, o d o r i ess , c r y s t a i iine powder.

PENICILLIN-G BENZATHINE

465

i.3. Compendiai References P e n i c i i i i n G benzathine i s i i s t e d i n t h e f o i i o w i n g compend i a : The European Pharmacopoeiai,

t h e U n i t e d S t a t e s Pharma-

copoeia w i t h t h e Code o f Federai Regulations' and t h e B r i t i s h Pharmacopoeia. 3

i .4. D e f i n i t i o n o f I n t e r n a t i o n a i U n i t One mg of pen c i i i i n G benzathine r e p r e s e n t s i 2 i i pen c i i i i n units. 7.5. Content The US Pharmacopoeia demands a c o n t e n t o f n o t i e s s t h a n 57.9 % and n o t more than 77.6 % of p e n i c i i i i n G and n o t i e s s than i 0 5 0 and n o t more t h a n i 2 7 2 U/mg. The European and B r i t i s h Pharmacopoeias r e f e r t o t h e substance w i t h o u t water. I t s h o u i d c o n t a i n a t i e a s t 96.0 % t o t a i p e n i c i i i i n and 24.0 t o 27.0 % benzathine. 2.

PRODUCT I ON

Szabo

Edwards and Bruce4 d e s c r i b e d a f r a c t i o n a t e d a d d i -

t i o n o f a s o i u t i o n o f benzathine a c e t a t e i n w a t e r t o a s o i u t i o n o f p e n i c i i i i n - N a i n water. When 60 % o f t h e benzat h i n e a c e t a t e i s added, t h e p r e c i p i t a t e i s f i i t e r e d and washed w i t h w a t e r . To t h e f i i t r a t e a r e added 35 % o f t h e benzathine a c e t a t e and t h e p r e c i p i t a t e t r e a t e d i i k e b e f o r e . F i n a i i y 5 % o f t h e benzathine a c e t a t e a r e added t o t h e fiit r a t e . T h i s f r a c t i o n a t i o n method y i e i d s a m i c r o c r i s t a i i i n e powder. Dropping b o t h m o i e t i e s simui t a n e o u s i y i n t o w a t e r , a p r o d u c t w i t h c r y s t a i s o f about i 0 0 um s i z e w i i i r e s u i t

/

a f t e r washing w i t h acetone, w a t e r and d r y i n g . When t h e p r e c i p i t a t i o n medi um c o n t a i n s formamide

the for-

466

FRANZ KREUZIG

mation o f needies i s prevented, which i s i m p o r t a n t f o r 5 p a r e n t e r a i appi i c a t i o n . F o r o r a i a d m i n i s t r a t i o n a product o f inhomogeneous c r y s t a i form i s o b t a i n e d by r e c r y s t a i i i s a t i o n from w a t e r and ace4 tone. 3.

PHYSICAL PROPERTIES

3.7. S o i u b i i i t y P e n i c i i i i n G benzathine i s p r a c t i c a i i y i n s o l u b i e i n c h i o r o form and e t h e r . The s o i u b i i i t y i n w a t e r i s a t h e r iow. 4 Szabo, Edwards and Bruce determined t h e so u b i i i t y i n f o r mamide, acetone , ethanoi , benzene and water a t d i f f e r e n t temperatures

.

Soi u b i 1 it y (mg/mi ) o f Peni c i i7 i n G Benzathi e i n Formami de/Water M i x t u r e s a t D i f f e r e n t Temperatures Formami de

[-%-7

273K( O°C)

298K( 25OC)

3 i 5K( 4 2 O C )

333K( 6OoC)

i00

2 i .2

28.0

92.5

7 58.0

95

3.2

3.8

72.0

43,O

90

3.2

3.2

i0.3

3 i .6

75

1.4

7.4

3.6

i3.2

50

7 .o

i .o

5.7

25

0.3

0.4

2.5 0.7

2.0

S o i u b i i i t y (mg/mi) o f P e n i c i i i i n G Benzathine i n D i f f e r e n t So7 vents a t D i f f e r e n t Temperatures Temp. [-K(OC)-i

acetone

ethanoi c')

benzene

water

qnc 1 1 9 \ LYO _. - I, -L- 3I 1

i c

3 i 3 (40)

3.8

16.2

0.55

0.i7

322 (49)

4.3

23.9

0.72

0.27

334 ( 6 i )

---

52.9

i. i o

0.46

I

,

I .3

3- ..L -

n

90

U . J.~ O

n i c

u.

13

467

PENICILLIN-G BENZATHINE

3.2. M e i t i n g Range A decomposition p o i n t o f 383 K (iiO°C) i s given6, t h e de-

termination o f the m e i t i n g point, according t o L. K o f l e r , g i v e s an i n t e r v a i from 385

-

388 K ('172

-

ii5OC)? Crystais,

embedded i n i drop of i i q u i d p a r a f f i n e , w i l l decompose between 403 and 409 K (130 and i36OC). Other authors

5

found 383

-

408 K (710 - i35OC).

3.3. O p t i c a i R o t a t i o n P e n i c i i i i n G benzathine i s d e x t r o r o t a t o r y : !'a-7i5

= t 206'

6 ( c = i , methanoi ) .

3.4. C r y s t a i P r o p e r t i e s These p r o p e r t i e s depend on t h e method o f p r e c i p i t a t i o n . The c r y s t a i s shouid have a shape so as t o be suspended e a s i i y i n a i i q u i d c a r r i e r f o r i n j e c t i o n . Adding forrnamide t o the i i q u i d f o r p r e c i p i t a t i o n prevents the formation o f needies and forms p i a t e - i i k e c r y s t a i s , which can be broken t o an average p a r t i c i e s i z e o f 750 urn. 5

I

A method f o r t h e d e t e r m i n a t i o n o f t h e f r i a b i i i t y ( i n g/sec) was given.8 The importance o f t h e a n g i e o f n a t u r a l s l o p e 9 ,

d i s p e r s i o n c h a r a c t e r i s t i c s , t h e cohesiveness and w e t t a b i i i t y have been discussed

i o.

3.5. U i t r a v i o i e t Spectrum

A spectrum of a m e t h a n o i i c s o l u t i o n o f p e n i c i l i i n G benzat h i n e (500 ug/mi), o b t a i n e d w i t h a Z e i s s DM 4 s p e c t r o -

I

photometer, i s shown i n f i g u r e i: t h e r e l e v a n t parameter i s t h e absorbance o f t h e s h o u l d e r a t 263 nm, which can be measured f o r spectrophotornetric assay.

250

Figure i : UV-Spectrum (see chapter 3.5.)

300nm

PENICILLIN-G BENZATHINE

469

3.6. Infrared Spectrum The infrared spectrum of p e n i c i i i i n G benzathine, obtained as a KBr-tabiet ( 1 . 2 mg i n 300 mg), was r u n on a Perkin Eimer i 7 7 . I t i s shown i n figure 2. The most s i g n i f i c a n t s t r e t c h i s t h a t a t i780 cm-l which is assigned t o the l3-iactam structure. 3.7. Nuciear Magnetic Resonance Spectrum The proton NMR spectrum was obtained i n DMF-d7 solution containing TMS as internal reference, u t i l i z i n g a Varian EM-390 NMR spectrometer operating a t a frequency of 90 MHz (see figure 3 ) . Chemicai s h i f t s

Muitipiicity

Intensity

Assignment

( 6 Y ppm)

8.60 7.30 5.60 5.50 4.20 4. i o 3.70 3.20 i .60 i .50

d ( J = 7 Hz)

m

iH i0 H

S

6H 2 H iH 2H 2 H

S

2 H

S

3H 3H

S

2 d S S

S

-CO-NHPhenyi -HtNH t H20 -2 H-5’ -6 H H -3 Phenyi -CH2-N Phenyi-CJ2-C0 N-CH -2 -CH -2 - N 2R-CH -3 2a-CH -3

d = doubiet, s = s i n g i e t , m = muitipiet, J = coupling constant

These data a r e in good aqreement w i t h the paper of Wiiii son e t a i . 3.8. Mass Spectrum The mass spectrum of p e n i c i i i i n G benzathine was run on a Varian MAT 3-11 in the f i e i d desorption mode (heating

470

W W v)

*----L-

1 .n

3+

,

I20

1

f-t- -

.-

I

,

-t

-

--

-

.... . .

.. . I

7 -

JUO

600

1

A END OF SWEEP

.

!

-!I

:. _c

Figure 3: NMR-Spectrum (see chapter 3 . 7 . )

CO

I

I

I 150

I 0 I

FRANZ KREUZIG

472

current i 0

-

20 mA). I n t e n s i v e i o n s w i t h t h e mass number

o f 5 7 5 can be a t t r i b u t e d t o p e n i c i i i i n G

+

benzathine + H+,

w h i i s t 9iO corresponds t o an a s s o c i a t e composed o f two peniciiiin G

4.

+

benzathine

+ Ht ( f i g u r e 4 ) .

STABILITY, DEGRADATION, ARTEFACTS P e n i c i i i i n G benzathine i s very s t a b i e because o f i t s iow s o i u b i i i t y i n w a t e r and b i o i o g i c a i media, r e s p e c t i v e i y . Being d i s s o i v e d i n aqueous media, t h e p e n i c i i i i n G moiety

wiii degrade i n a r a t h e r compiex manneri2, i3 e l u c i d a t e d by NMR s t u d i e s .

which c o u i d be

These degradation p a t t e r n s may be compiicated by t h e aminoi y s i s o f p e n i c i i i i n G by t h e benzathine c a t i o n . I t c o u i d be demonstrated t h a t t h e r e a c t i o n of i,Z-diami noethane (monocation) w i t h p e n i c i i i i n G g i v e s an increased r e a c t i o n r a t e compared w i t h t h e monoamine o f s i m i i a r b a s i c i t y . This i s a t t r i b u t e d t o i n t r a m o i e c u i a r generai a c i d c a t a i y s i s which i n t u r n i n d i c a t e d t h a t n u c i e o p h i i i c a t t a c k takes p i a c e from t h e l e a s t hindered a - s i d e i n disagreement w i t h i4 t h e p r e d i c t i o n o f t h e t h e o r y o f s t e r e c e i e c t r o n i c control:

5.

BIOPHARMACEUTICS

5. i Pharmacokinetics

Because o f i t s iow s o i u b i i i t y i n w a t e r p e n i c i i i i n G benzathine i s the iongest acting p e n i c i i i i n ; the resultinq c o n c e n t r a t i o n i n serum and u r i n e r e s p e c t i v e i y are, o f course, r a t h e r low. On t h e o t h e r hand t h i s drug o f f e r s , according t o t h e amount administered, t h e r a p e u t i c p e n i c i i i i n G i e v e i s f o r about one month.

.

I-

I

I00

P w

I Y Q:

50

W

L I-

4

w

a

500

600

700

MASS NUMBERS

Figure 4 : Mass-Spectrum (see chapter 3 . 8 . )

800

FRANZ KREUZIG

414

Peni c i iii n G benzathi ne, administered intramuscui a r i y , i s s u i t e d f o r t h e p r o p h y i a x i s and l o n g term therapy o f syphii i s and rheumatic diseases. E i i a s e t aiei5 f o l i o w e d serum p e n i c i i i i n i e v e l s i n man a f t e r i n j e c t i o n o f t h i s compound, and conciuded t h a t i t showed no s i g n i f i c a n t t o x i c i t y and gave serum p e n i c i i i i n i e v e i s o f g r e a t e r d u r a t i o n than any reported w i t h o t h e r forms o f r e p o s i t o r y peni c i i1i n s . When 2.400.000 I U o f p e n i c i i i i n G benzathine are i n j e c t e d , the bioassay showed t h e existence of mean l e v e l s above 0.036 I U / m i serum and above 20 I U / m i u r i n e f o r t h e d u r a t i o n o f 26 days. The corresponding values f o r one hour a f t e r 16 t h e i n j e c t i o n were 0.3 and 234 I U / m l r e s p e c t i v e l y . The i n j e c t i o n o f p e n i c i i i i n G benzathine suspensions may cause pain, t h e r e f o r e i o c a i a n e s t h e t i c s are added. 5.2. Metaboi ism The p e n i c i i i i n G moiety of t h e drug decomposes i n p r i n c i pa7 as mentioned i n chapter 4.

6.

METHODS OF ANALYSIS

6.i. Identification The sampiei

y3

i s shaken w i t h 0.1 N NaOH, e x t r a c t e d w i t h

ether, evaporated, t h e residue i s d i s s o i v e d i n ethanoi. A f t e r the addi t i on o f p i c r i c acid, h e a t i ng and cooi ing , the p r e c i p i t a t e d c r y s t a i s a r e separated; t h e i r me7 ti ng p o i n t i s 487 Simoesi7

K (2i4OC).

d i s s o i v e d t h e sampie i n 0.2

M NaOH and e x t r a c t e d

t h e benzathine moiety w i t h e t h e r . A f t e r evaporating t h e residue, H C i (-10 % ) and NaN03 (10 % ) are added, t h e mix-

PENICILLIN-G BENZATHINE

415

t u r e i s e x t r a c t e d w i t h e t h e r , t h e e t h e r i s evaporated and t h e r e s i d u e mixed w i t h H2S04. A green c o i o r i s observed. A d d i t i o n o f w a t e r g i v e s a p i n k c o i o r , a f t e r adding NaOH t h e c o i o r changes t o b i u e . 6.2.

Iodometric T i t r a t i o n The f i r s t method was d e s c r i b e d by Aiicinoi8.

Underivatized

p e n i c i i i i n i s i n e r t t o i o d i n e i n n e u t r a i aqueous s o i u t i o n . A f t e r h y d r o i y s i s w i t h a7 k a i i , t h e r e s u i t i n g p r o d u c t s consume 8 t o 9 moies o f i o d i n e . The d i f f e r e n c e o f t h e i o d i n e consumption b e f o r e and a f t e r h y d r o i y s i s i s p r o p o r t i o n a i t o t h e q u a n t i t y o f p e n i c i i i i n . P e n i c i i i i n G consumes 8.97 e q u i v a i e n t s o f i o d i n e p e r moi; t h i s r a t i o depends on t h e c o n d i t i o n s o f assay, t h e r e f o r e a b i i n d sampie has t o be taken i n t o account. The h y d r o i y s i s t i m e i s i 5

-

30 m i n u t e s .

Ei-Sebai e t a i . i 9 d e s c r i b e d t h e a n a i y s i s o f p e n i c i i i i n G benzathine i n 0.067 M Na2HP04 s o i u t i o n w i t h JCi i n H C i s o i u t i o n ; t h e p e n i c i i i i n l i b e r a t e d was t i t r a t e d w i t h 0.05 M KJOg i n CHCi3;

t h e e n d - p o i n t was t h e disappearance

o f t h e c o i o r f r o m t h e CHCi3-iayer. The i n f i u e n c e o f i o d i n e consuming i m p u r i t i e s a r e discussed by L e B e i i e e t a i . 2 0 who proposed a m o d i f i c a t i o n o f t h e i o d o m e t r i c method and found t h a t benzathine and i o d i n e 2i form a benzimidazoiinium s a i t

which i n t e r f e r e s i n t h e i o d o m e t r i c assay.

FRANZ KREUZIG

476

6.3. Nonaqueous T i t r a t i o n F o r t h e d e t e r m i n a t i o n o f t h e b e n z a t h i n e m o i e t y 3 t h e substance i s d i s s o i v e d i n a s o i u t i o n o f NaCi and NaOH, ext r a c t e d w i t h e t h e r , t h e combined e x t r a c t i s washed w i t h water, t h e combined washings a r e e x t r a c t e d w i t h e t h e r , t h e e x t r a c t s a r e evaporated t o dryness, t h e r e s i d u e i s d i s s o i v e d i n anhydrous g i a c i a i a c e t i c a c i d and t i t r a t e d w i t h 0 . i M HCi04, u s i n g i - n a p h t h o i - b e n z e i n as i n d i c a t o r ; t h e o p e r a t i o n i s repeated w i t h o u t t h e substance t o be t e s t e d ( b i ank v a i ue)

.

i m i o f 0 . i M HCi04 i s e q u i v a l e n t t o O.Oi202 g o f benzathine. 6.4. Spectrophotometri c Assay P e n i c i i i i n G i n p e n i c i i i i n G benzathine i s determined by measuring t h e e x t i n c t i o n o f a s o i u t i o n o f 50 mg o f sampie i n -100 m i a b s o l u t e methanoi a t 263 nm i n comparison t o a r e f e r e n c e s t a n d a r d2 . HoibrookZ2 determined p e n i c i i i i n G b e n z a t h i n e a f t e r heat i n g t h e substance w i t h a Na-acetate/CH3COOH s o i u t i o n (pH = 4 . 6 ) , c o n t a i n i n g a t r a c e of CuS04. The absorbance o f t h e r e s u i t i n g p e n i c i i i i n a c i d i s measured. The method

has a s t a n d a r d e r r o r o f 3 % and i s a p p i i c a b i e t o preparat i o n s w i t h a potency of a t l e a s t 0.5 U/rng. I t has been found t h a t benzathine forms a w a t e r s o i u b i e

compiex w i t h B i e b r i c h ~ c a r i e t ~The ~ . absorbance i s measur e d a t 5-16 nm and i s r e l a t e d t o t h e p e n i c i i i i n G benzat h i n e c o n t e n t . The same a u t h o r s 2 4 s t a t e d t h a t p e n i c i i i i n forms a compiex w i t h methyiene b i u e , which i s measured a t 655 nm and shows c o r r e l a t i o n w i t h t h e p e n i c i i i i n G b e n z a t h i ne c o n t e n t .

PENICILLIN-G BENZATHINE

477

For the determination o f p e n i c i i i i n G benzathine i n o i n t ments t h e r e a c t i o n w i t h n i t r o p r u s s i de-Na and KMn0424 i n a i k a i i n e s o i u t i o n produces a b i u e c o i o r . The s e n s i t i v i t y i s 2000 U / m i o f ointment. 6.5. M i c r o b i o i o g i c a i Assay

The sampie i s d i s s o i v e d i n methanoi and f u r t h e r d i l u t e d w i t h i % phosphate b u f f e r pH = 6.4 so as t o g i v e a conc e n t r a t i o n o f i . 0 U o f p e n i c i i i i n G/m7. The a g a r d i f f u s i o n assay i s performed w i t h Staphylococcus aureus (ATCC 29727)*.

6.6. Thin-Layer Chromatography

Layer i.

ceiiuiose

Soivent Systems

V i suai ization

CH30H/2-butanoi/CHCi 3/HCi 70 % = iO/iO/iO/i

2. siiica gei + CH30H/i-butanoiiCHCi3/CH3COOH cei i ui ose = iO/lO/iO/i si i i ca gei

=

=

Ref

J2

25

J2

26

butyi acetate/CH3COOH/MeOH/i-butanoi / phosphate buffer pH=7.3 = 80/4/5/i 5/20

FeCi3 + K3!-Fe(CN)6-7

+ HCi

27

4. siiica gei

acetone/HCOOH

FeCi3 + K3!-Fe(CN)6-7

+ H2S04

28

5. siiica gei

isoamyi acetate/CH30H/HCOOH/H20 (upper phase) = 65/20/i0/5

NaN02 + NH4 suifamate + N-( i-naphthyi )ethyiendiamine

28

6. siiica gel

CH3COOH/ butyi acetatel 0.i M phosphate buffer pH=5.6/i-butanoi/CH30H = = 20/40/i2/5/7,5

KJ + H2PtCi6

- acetone

29

3.

=

95/5

-1.

HCi

PENICILLIN-G BENZATHINE

479

Comments : ad 1:

The d e t e c t i o n l i m i t s were i n t h e range o f 5-30,ug, i t was s t a t e d , t h a t c e i i u i o s e has advantages o v e r

s i i i c a gei. ad 2:

The a u t h o r s c l a i m t h e ease o f t h e p r o d u c t i o n o f t h i s p i a t e and t h e s p e c i f i t y o f t h e c o l o r o f t h e spots a f t e r s p r a y i n g .

ad 3:

With t h i s method i m p u r i t i e s and decomposition p r o ducts were i d e n t i f i e d and q u a n t i t a t e d v i s u a l l y .

ad 4 and 5: Aithough t h i s method i s used f o r p e n i c i i i i n G proc a i n , i t r e f e r s t o t h e decomposition o f p e n i c i i i in G i n aqueous s o l u t i o n s t o benzyi peni c i iiic a c i d and b e n z y i p e n i c i i i o i c a c i d . F u r t h e r decompos i t i o n o f p e n i c i i i i n G occurs when a i c o h o l i c soi u t i o n s a r e used f o r s p o t t i n g t h e chromatograms, t h e corresponding a1 k y i -a-D-peni c i i io a t e b e i ng formed. The usefuiness o f d i f f e r e n t spray reagents i s discussed. ad 6:

T h i s method a l i o w s f o r t h e d e t e c t i o n of p e n i i i o i c a c i d and p e n i c i i i o i c a c i d amongst t h e main components o f p e n i c i i i i n G benzathine.

6.7. High Performance L i q u i d Chromatography The paper o f L e B e i i e e t a i .30, c o n c e r n i n g t h e HPLC d e t e r m i n a t i o n o f p e n i c i i i i n V benzathine, enabies t h e separat i o n o f p e n i c i i i i n G from benzathine too, so i t can be used f o r t h e i n d i r e c t d e t e r m i n a t i o n o f p e n i c i i i i n G benz a t h i n e . The compounds a r e s e p a r a t e d on a RP 8-coiumn w i t h t h e e i u e n t CH30H/0.05

M phosphate b u f f e r pH=3.5

=

53/47, t-h-e UV-detector was s e t t o 274 nm. Nachtmann and G s t r e i n ” have m o d i f i e d t h i s method i n t h e f o i i o w i n g

FRANZ KREUZIG

480

aspects : Eiuent: CH30H/phosphate b u f f e r 0.06 M t 7 % t r i e t h y i a m i n e pH= 5.5 = 30/70, T = 323 K ( 5OoC) , h = 220 nm. With i n c r e a s i n g pH o f t h e e i u e n t t h e r e t e n t i o n time o f benzathine increases, This method o f f e r s t h e p o s s i b i i i t y o f the q u a n t i t a t i v e determination o f both m o i e t i e s o f t h e moi ecui e. Puttemans e t

have shown t h a t t h e e i u t i o n o r d e r o f

benzathine and p e n i c i i i i n can be changed by means o f ionp a i r HPLC, a p p i y i ng t h e e i u e n t CH30H/phosphate b u f f e r 4 i 0 M tetrabutyiammoniumpH=3.0 = 30/70, adding 5 b romi de

7.

.

.

ACKNOWLEDGMENTS I am indebted t o D r . B. Prager, Biochemie GmbH, Kundi ,

f o r performing and i n t e r p r e t i n g t h e NMR-spectrum, t o D r . G. Schuiz, Sandoz F o r s c h u n g s i n s t i t u t GmbH, Wien, and

D r . A. N i k i f o r o v , Organisch-Chemisches I n s t i t u t der Univ e r s i t a t Wien, f o r performing and i n t e r p r e t i n g t h e massspectrum.

a.

REFERENCES

7 ) European Pharmacopoeia, Council o f Europe, I 1 1

-

i975

2) The United States Pharmacopoeia, Twentieth Revision, 7980, w i t h t h e N a t i o n a i Formulary, F i f t e e n t h E d i t i o n , -1980, United States Pharmacopoeia7 Convention, I n c .

3) B r i t i s h Pharmacopoeia 7980, London, Her M a j e s t y ' s S t a t i o n e r y O f f i c e , 7980

4 ) J.L. Szabo, C.D.

Edwards and F.W. Bruce, i n R. Brunner,

G. Machek: Die A n t i b i o t i k a , i/i Aiigemeiner T e i i

-

PENICILLIN-G BENZATHINE

48 1

P e n i c i i i i n , V e r i a g Hans C a r i , Nurnberg, 7962, p. 344 5 ) D.P. 947 826 6 ) J.L.

Szabo, C.D.

G.Machek:

Edwards and F.W. Bruce, i n R. Brunner,

D i e A n t i b i o t i c a , i/i A i i g e m e i n e r T e i l -

P e n i c i i i i n , V e r i a g Hans C a r i , Nurnberg, 7962, p . 259 7) U s t e r r e i c h i s c h e s Arzneibuch, 9. Ausgabe, 11. Band, U s t e r r e i c h i s c h e S t a a t s d r u c k e r e i , Wien, i 9 6 9 , p . 376 8 ) M.L. E z e r s k i i : Khim.-Farm.

4, 54 (7970) Zh. -

9 ) M.L. E z e r s k i i : Khim.-Farm.

4, 47 (1970) Zh. -

i 0 ) M.L. E z e r s k i i , G.M. Pismennaya: Khim.-Farm.

__

Zh. 9, 9 i

( i 976)

1 1 ) W.L.

W i i s o n , H.W. A v d o v i c h and D.W. Hughes: J. AOAC 57,

i300 (i974) i 2 ) M.A. Schwartz and F.H.

B u c k w a i t e r : J. Pharm. S c i . 5i,

i i i 9 (i962) i 3 ) F. M i t s u m o r i , Y . A r a t a , S. F u j i w a r a , M. Muranaka and Y . H o r i u c h i : B u i i . Chem. SOC. Jap. 50, 3764 ( i 9 7 7 )

i 4 ) A.F. M a r t i n , J.J. M o r r i s and M . I .

Page: J.C.S.

Chem.

6, 298 ( i 9 7 9 ) Comm. i 5 ) W. E i i a s , A.H.

P r i c e and H.J. M e r r i o n : A n t i b i o t . and

Chemother. i, 4 9 i ( i 9 5 i )

i 6 ) H. Fiamm, A. Luger, F. P a v i i k and M. R o t t e r : Wien. 88, 384 (1976) K i i n . Wschr. i 7 ) R. Simoes, i n Vogei: Textbook o f P r a c t i c a i O r g a n i c Chemistry , Longmans, London , 7 948 , p. 627 i 8 ) J. A i i c i n o : I n d . Eng. Chem., A n a i . Ed. i8, 6 i 9 (i946) 79) I. E i - S e b a i , S.M.

Rida, Y.A.

B e i t a g y , M.M.A.

Ei-Khaiek:

Pharmazie 29, i 4 3 (1974) 20) M.J.

L e B e i i e and W.L. W i i s o n : J. Pharm. S c i . 67, 1495

( i 978) 27) M.J. L e B e i i e and W.L.

W i l s o n : J. Pharm. S c i . 68, i 3 2 2 ,

( i 979) 22) A. H o i b r o o k : J. Pharm. Pharmacoi. i 0 , 762 ( i 9 5 8 )

FRANZ KREUZIG

482

23) H.M.

Nour Ei-Din, Y.M.

Dessouky, M. E l - K i r d a n i : B u l l .

Fac. Pharm., Cairo Univ., i 0 , 779 (797'1)

24) G. Miihaud, L. P i n a u i t , J.P. Moretain: Am. F a i s i f . 68, i 9 i (1975) Expert. Chim. 25) G.S. Chung, R.T.

Wang Tai-Wan K ' o Hsue, 27, 27 (1973)

26) G.S.

Wang

Chung, R.T.

Hua Hsueh 4, 122 (7977)

27) J.J. Cruceanu, M. Med anu, Zen t r a 7 b 7 a t t P ha r m 776, -

.

E. Aiteanu and

A. Moldovan:

Pha rmak o t he r . Labo r a t o riums d ia g n .

25i (1977)

28) J.R. Fooks and G.L. Mattok: J. Pharm. Sci 58, i357

( i969) 29) W.J. Wilson, M.J.

L e B e i l e and K.G

Graham: J. Pharm.

Sci. i 4 , 27 (7979) 30) M. LeBeile,

K. Graham and W.J. W i son: J. Pharm. S c i .

68, 555 (7979) 3i)

F. Nachtmann and K. G s t r e i n : J. Chromatogr., submitted f o r pubiication

32) M. Puttemans, L. Dryon and D.L. Massart: J . Chromatogr., i n press

PHENYLBUTAZONFI Syed Laik Ali

History Description 2.1 Name, Formula, Molecular Weight 2.2 Appearance, Colour, Odour 3. Synthesis 4. Physical Properties 4.1 Melting Range 4.2 Solubility 4.3 Dissociation Constant 4.4 Loss on Drying 4.5 Ultraviolet Spectrum 4.6 Infrared Spectrum 4.7 Nuclear Magnetic Resonance Spectrum 4.8 Mass Spectrum 4.9 Differential Scanning Calorimetry (DSC), Differential Thermal Analysis (DTA), Thermogravimetry (TG) 4.10 X-Ray Diffraction Spectrometry 4.11 Scanning Electron Microscopy Colour Reactions 5. 6. Degradation and Stability 7. Dissolution 8. Methods of Analysis Drug Metabolism and Pharmacokinetics 9. References 1. 2.

Analylical Profiles of Drug Subslances Volume I I

483

484 484 484 484 484 486 486 486 487 487 487 487 490 490 493

497 499 499 502 507 508 516 518

Copyright 0 1982 by The American PharmaceuticalAssociation

ISBN 0-12-260811-9

SYED LAIK ALI

484

1. History After second world war a German Chemist Hans Stenzl synthesised phenylbutazone, 1,2-diphenyl4-n-butylpyrazolidin-3,5-dion in the research laboratories of J.R. Geigy in Basel, Switzerland (1). This compound formed water-soluble salts and was used in combination with aminophenazone in injection solutions. Phenylbutazone was brought in the market in 1952 as a single preparation under the trade-name of Butazolidin. It has remarkable antirheumatic and antiphlogistic properties.

2. Descr iption 2.1. Name, Formula, Molecular Weight 4-Butyl-1,2-diphenyl-3,S-pyrazolidinedione C19H20N202

308.38

I 2.2. Appearance, Colour, Odour A white or almost white, crystalline powder, practically odourless, with a slightly bitter taste

.

3. Syn thesi s The first synthesis was carried out by heatincl n-butyl diethyl malonate with hydrazobenzene and sodium alcoholate ( 3 ) .

485

PHENY LBUTAZONE

n-CCLHS-CH-COOC2Hg

I

COOC2H5

+

HN-c&

I

HN-C6H5

Another method of synthesising phenylbutazone (3) consists of reacting diethyl malonate with hydrazobenzene which is further treated with crotonaldehyde and subsequently hydrogenated catalytically.

I

SYED LAIK ALI

486

I C6H5

H2

I C6H5 4. Physical Properties 4.1.Melting Range (4): Phenylbutazone melts between 104 and 107OC.

4.2. Solubility (5) : The solubility o f phenylbutazone in various solvents at 2 0 0 ~is given in Table 1 as 1 part per specified parts-solvent. TABLE 1 Solubility of Phenylbutazone at 2OoC (5) Solvent Water Ethanol Methanol Ether Chloroform Aceton Benzene

Solubility practically insoluble 28 parts 18 parts 15 parts 1.3 parts 2.5 parts 3.5 parts

PHENYLBUTAZONE

481

4.3. D i s s o c i a t i o n C o n s t a n t P h e n y l b u t a z o n e h a s a n a c i d i c h y d r o g e n atom a t C 4 . The P k a d e t e r m i n e d i s r e p o r t e d t o be 4.89 (6). P h e n y l b u t a z o n e is c o n s i d e r e d a c a r b o n a c i d (carbon acids are acids i n which t h e d i s s o c i a t i n g p r o t o n is bound t o a c a r b o n atom i n s t e a d of h e t r o atoms s u c h a s oxygen or n i t r o g e n ) a n d t h e P k a v a l u e b e t w e e n 4.5 - 4.7 h a s a l s o b e e n g i v e n ( 7 , l l ) . The d e p e n d e n c e o f Pka v a l u e s o f p h e n y l b u t a z o n e on t h e s o l v e n t medium h a s a l s o b e e n reported ( 8 ) . Pka v a l u e s i n pure m e t h a n o l , e t h a n o l and water a r e g i v e n a s 5.42, 5.76 a n d 5.07 respectively (8). 4.4. Loss on When d r i e d i n o f 30 2 1 0 mm more t h a n 0.5

Dryin vacuu: a t 8OoC a n d a t a P r e s s u r e o f m e r c u r y f o r 4 h o u r s ii looses n o t % of i t s w e i g h t ( 9 ) .

4.5. U l t r a v i o l e t Spectrum (10) Phenylbutazone i n s o l u t i o n absorbs u l t r a v i o l e t r a d i a t i o n to produce a spectrum w i t h d i f f e r e n t maxima i n d i f f e r e n t s o l u t i o n s . I n m e t h a n o l , 0 . 1 N HC1 a n d 0 . 1 N NaOH i t h a s a b s o r p t i o n maxima a t 243, 235 a n d 263 nm r e s p e c t i v e l y . The corres o n d i n g molecular e x t i n c t i o n c o e f f i c i e n t s a n d Ale,, a r e r e p o r t e d t o be ( 1 0 ) :

!?%

Methanol Absorption Maximum A%

A I'wl €

0 . 1 N HC1O.l N N a O H

243 nm

235 nm

263 nm

482

440

669

14860

13570

20630

The U V s p e c t r a a r e g i v e n i n F i g . 1. T h e a b s o r p t i o n maxima i n a c i d a n d a l k a l i n e s o l u t i o n s a t 232 nm a n d 264 nm h a v e a l s o b e e n r e p o r t e d (37). 4.6. I n f r a r e d Spectrum (10,11,62) The i n f r a r e d spectrum o f p h e n y l b u t a z o n e is g i v e n i n F i g . 2 . The spectrum was o b t a i n e d w i t h a P e r k i n - E l m e r 257 s p e c t r o p h o t o m e t e r from a KBr p e l l e t . T h e r e is a good a g r e e m e n t w i t h t h e s p e c t r a r e p o r t e d i n l i t e r a t u r e ( 1 0 , 1 1 , 6 2 ) . The s t r u c t u r a l

YO

F i g . 1.

UV Spectrum of P h e n l y b u t a z o n e

--- ( ) .1N NaOH) , - - (0.1N HCL)

488

in

-(Methanol),

Microns

25

3a

50

Lo

60

70

03 P

W

-r

(01- 1 1

F i g . 2. I R Spectrum of P h e n l y b u t a z o n e , KBr P e l l e t , P e r k i n E l m e r 257 S p e c t r o p h o t o m e t e r

SYED LAIK ALI

490

assignments may be correlated with the following band frequencies

.

Frequency (cm-1 1720 and 1755

Assignment Characteristic stretching vibrations of C = 0 group. Characteristic skeletal stretching vibrations of the aromatic ring. Characteristic band of dioxopyrazolidine compounds. Bands of monosubstituted phenyl.

)

1600 and 1490 1300 760

-

695

4.7. Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectrum of phenylbutazone as shown in Fig. 3 was obtained on a Varian T-60 NMR spectrometer in deuterated chloroform containing 1 % tetramethylsilane as the internal standard. The following spectral assignments are made for Fig. 3: Chemical Shift ( &

-

)

Assignment

0.95 2 ppm doublet and multiplet

Protons of n-butyl rest.

3.35 ppm triplet

Proton at C

7.30 ppm mu1 t iple t

Aromatic phenyl protons.

-

4 position.

There is a good agreement with the reported values in literature (62). 4.8.

Mass Spectrum (121 Mass spectrum is given in Fig. 4 Instrument: Varian CH5 Sample temperature (direct inlet): 8OoC Source temperature: 18OOC Electron energy: 70 eV

The prominent ions of this spectrum can be correlated to the structure as following: m/e 308 = M 183 = C6H5NHNC6H5 265 = M C3H7 105 = CgHtjN2 252 = M C4H8 77 = CgH5

-

..I

..

I , . . . l . . . . 1 . . . * l , * ,

I . .

8.0 Fig.

7.0

3.

. .

1 . .

61)

,

, ' i ., . , " i . . " . i ~ " . ~ i ~ ' ~ ~ . i 1 . ' ~ * ' 5.0 m(6) 6.0 3.0 20 I .o 0

N M R Spectrum of Phenlybutazone, Varian T60 Spectrometer.

I

103

0 10: 70eVV1180.C. Diddinlet: 8O'C

Fig. 4.

Mass spectrum of P h e n l y b u t a z o n e

PHENY LBUTAZONE

493

The elemental compositions of these ions have been determined by high resolution mass spectrometry (instrument: CEC 21 - 110) and are in agreement with the assignments made above (12). The fragmentation pathways of Phenylbutazone and oxyphenbutazone were established by means of deuterium labeling, metastable peaks and accurate mass determinations. The major pathways are the McLafferty rearrangement of the molecular ion and formation of azobenzene and substituted azobenzene ions (13). The mass spectra of the methyl derivatives have also been discussed (13,14). 4.9. Differential Scanning Calorimetry (DSC), Differential Thermal Analysis (DTA), Thermogravimetry (TG) The solid-solid transition of phenylbutazone polymorphs by heating was investigated by DSC and DTA (15). Three polymorphs of phenylbutazone were observed. Form I melted at 103O, Form I1 twice at 93O and 103O and Form I11 at 93O (15). Form I1 melted to an opaque paste at 93O, solidifying immediately and melting again at 103OC. Furthermore, the mutual transition phenomenon among these three polymorphs was observed by heating through DSC (15). In another investigation solid-solid and solid-liquid transitions of phenylbutazone polymorphs were investigated by DSC, DTA and TG (16). Four polymorphs were observed with transition points at 93.4, 95.1, 106.0 and 107.5OC (16). The curves are reproduced in Fig. 5. In addition two pseudopolymorphs from cyclohexane and isobutanol were determined containing one mole solvent to three moles phenylbutazone. The mutual transition behaviour of the polymorphs was investigated, the melting enthalpies determined and the energy of activation calculated from DSC-data. The DSC-curves of the different crystalline modifications were recorded on a Dupont 990 thermal analyzer, equipped with a DSC-cell 910 and calibrated with Indium (16). Thermal data of phenylbutazone polymorphs is given in Table 2, DSC and DTA/TG curves are reproduced in Fig. 6 (16).

494

SYED LAIK ALI

MODIFICATION

if i f

2- PROPANOL

RECRYST. FUSION OF

80

4

90

100

T(YC

F i g . 5. DSC-curves of p o l y m o r p h i c forms of p h e n l y b u t a z o n e and t h e i r m i x t u r e s o b t a i n e d by c r y s t a l l i z a t i o n f r o m d i f f e r e n t s o l v e n t s ( h e a t i n g r a t e 10°C/min, s e n s i t i v i t y 0.5 mcal/s i n , sample w e i g h t 2-3 mg).

PHENYLBUTAZONE

495

I \

a) from cyclohcxane; b) from isohutanol

TG7

cycloherane

Fig. 6. solva tes

Simultaneous DTA/TG-curves of phenylbutazone

SYED LAIK ALI

496

TABLE 2 Thermal Data of Phenylbutazone Polymorphs (16) Polymorph

w

Transition temperature

OC

79.3 71.1

93.4 95.1 106.0 107.5

P

t

b

Enthalpy of transition J/9

-

72.3

The thermal behaviour of the polyrnorphs under different treatment conditions has also been investigated (17). Compression of the thermodynamically unstable forms at a*compression force of 1590 - 2040 Kg induced polymorphic changes in the crystals. Similar changes were also produced through grinding. The apparent equilibrium solubilities of 4 polymorphs at their transition temperatures were determined (17). In Table 3 different transition temperatures and peak solubilities of the different polymorphs are given

.

TABLE 3 (17)

Differential Scanning Transition Temperatures and Peak Solubilities of the Different Polymorphs (171 Form

Solvent O F Crystallization

Transition Temperature

Peak Solubility in Phosphate buffer, pH 6.95, at 36OC,

mg/100 ml I I1 I11 IV

Isobuty 1 alcohol Cycloh e xane n- Heptane 2- Propano1-Water

-

8OoC

288.7

90%

279.9

93% 1050C

233.6 213.0

491

PHENY LBUTAZONE

Polymorphism o f p h e n y l b u t a z o n e b y a s p r a y - d r y i n g method h a s a l s o b e e n i n v e s t i g a t e d r e c e n t l y ( 1 8 ) .

DTA-curves o f a s p r a y - d r i e d p h e n y l b u t a z o n e from 5 % methylene c h l o r i d e s o l u t i o n are given i n Fig. 7 (18) The i n v e s t i g a t i o n o f a r e f e r e n c e s u b s t a n c e o f p h e n y l b u t a z o n e t h r o u g h DSC i n a c l o s e d s y s t e m g a v e following results (19): Impur i t i e s Melting p o i n t L a t e n t h e a t of f u s i o n

0.2 - 0 . 1 Mol% 105.5OC 6. 8 K cals./Mol

4.10. X-Ray D i f f r a c t i o n S p e c t r o m e t r y The c r y s t a l s t r u c t u r e o f p h e n y l b u t a z o n e h a s b e e n d e t e r m i n e d w i t h x - r a y d i f f r a c t i o n method ( 2 0 , 2 1 1 . T h i n n e e d l e - l i k e c r y s t a l s o f p h e n y l b u t a z o n e , obt a i n e d a f t e r c o n t r o l l e d e v a p o r a t i o n of a n a l c o h o l i c s o l u t i o n were u s e d f o r measurements ( 2 1 ) . The u n i t c e l l d i m e n s i o n s o f t h e c r y s t a l s were d e t e r m i n e d from o s c i l l a t i o n s a nd W e i s s e n b e r g p h o t o g r a p h s t a k e n about c r y s t a l l o g r a p h i c a x e s u s i n g n i c k e l - f i l t e r e d c o p p e r r a d i a t i o n . The c e l l d i m e n s i o n s o f t h e c r y s t a l s o f p h e n y l b u t a z o n e were r e f i n e d o n a comp u t e r - c o n t r o l l e d H i l g e r a n d Watts 4-circle d i f f r a c t o m e t e r . The c r y s t a l d a t a for p h e n y l b u t a z o n e i s given i n Table 4 (21). TABLE 4 C r y s t a l Data f o r P h e n y l b u t a z o n e ( 2 1 1 Space group p21/ c 21.695 + a i n Ao 5.823 T b i n Ao 27.861 7 c i n Ao i n degrees 108. 06 Volume o f t h e u n i t cell i n ~ 0 3 3348.565 Molecular f o r m u l a ClgHlg02Nz Form u l a we i g h t 307.168 No. o f f o r m u l a w e i g h t s 8 in the unit cell Measured d e n s i t y i n gm/cc 1.211 + Calculated density i n gm/cc 1.218

0.004 0.002 0.004

0.10

0.020

498

SYED LAIK ALI

90

100

110

Fig. 7 . DTA-curves of s p r a y - c r i e d phenylbutazone o b t a i n e d a t v a r i o u s d r y i n g t e m p e r a t u r e s . Numbers i n t h e f i g u r e i n d i c a t e d r y i n g t e m p e r a t u r e . A b s c i s s a : t e m p e r a t u r e , OC.

PHENY LBUTAZONE

499

Crystal structures of phenylbutazone and a 2:l complex between phenylbutazone and piperazine are also reported (22). X-ray powder diffraction patterns of phenylbutazone polymorphs and phenylbu tazone solvates have been also investigated (16). The diffraction patterns of three modifications are given in Fig. 8 (16). X-ray diffraction patterns of the three peaks of phenylbutazone polymorphs are given in Table 5 (16). TABLE 5 X-ray diffraction D a 9f the Dhenvlbutazone PolvmorDhs (16) Polymorph

4

B

02 theta 8.7 + 0 . 2 19.46.9 8.35 20.2 7.05 7.9 20.7 7.05

+

D

1/10

100 70 60 100 70

60

+ 0.2

100 70 60

4.11. Scanning Electron Microscopy (16,17) Photomicrographs were made using a Jeol JSM - U3 scanning electron microscope. Prior to investigations the substances were coated with gold using a direct coating sputter technique giving a coating layer of about 500 Ao. A photograph of polymorph modification is given in Fig. 9 (16). 5 . Colour Reactions When phenylbutazone is heated with glacial acetic acid and hydrochloric acid, hydrazobenzene is formed immediately which rearranges itself to benzidine. The blue or violet colour yielded is due to oxidation of hydrazobenzene. After addition of sodium nitrite and pouring into a solution of sodium carbonate and R-naphthol a red product is formed, which mainly consists of 4,4'-bis (2-hydroxyl-1-azonaphthyl) biphenyl and 2-hydroxynaphthyl1-azobenzene (23). Red colouration is produced when

SYED LAIK ALI

L ;I, i;.1

Fig. 8.

MODlFlKATlONd

Powder X-ray diffraction of phenlybutazone polymorphs.

PHENY LBUTAZONE

501

Fig. 9 Scanning Elektron Photomicrograph of Phenylbut azon ; Polymorph

s

magn. 1200 x

SYED LAIK ALI

502

sulfuric acid and potassium nitrate are treated with phenylbutazone which turns to orange-red on reaction with ammonia solution (24). Vanadium-sulfuric acid solution reacts with phenylbutazone to give a dark green colouration (25). An alcoholic solution of iron (111) chloride and 2,2'-dipyridyl reacts with phenylbutazone to give a red colouration (26). Hyrolysis of drug with phosphoric acid followed by the diazotization and coupling of the resulting products with R-Naphtol gives a colour reaction (27). 6 . Degradation and Stability Aqueous solutions of phenylbutazone sodium decompose in course of hydrolysis and oxidation, for example with sodium hydroxide and hydrogen peroxide (28,29) Degradation occurs also in oxygen-free basic solutions and in oxygenated solutions (30,31). Degradation pathways in other solvents such as N, N-dimethylformamide, N, N-dimethylacetamide, diethyl carbonate and propylene glycol-water have also been described (32). Four mainly occuring decomposition products of phenylbutazone are the following (29):

.

1. N- (2-Carboxycaproyl)-hydrazobenzene (Car-

boxylic acid of phenylbutazone) (I) 2. 4-Hydroxyphenylbutazone (11) 3 . N-(2-Carboxy-2-hydroxycaproyl)-hydrazoben-

zene (0(-hydroxycarboxylic acid of Phenylbutazone) ( 1 1 1 ) 4. n-Caproylhydrazobenzene (IV)

503

PHENY LBUTAZONE

C6H5\

0

C6 H5

N-N

0

9 HO

0 pC4HCJ

I1 C

C H5

\

N-N O\COOH

-n-CbHg

HO

I11 The oxidation product of phenylbutazone sodium in aqueous solution is 4-hydroxyphenylbutazone (11), the product of hydrolysis is N-(2-carboxcaproyl)hydrazobenzene (I). Under the action of hydroxyl ions on 4-hydroxyphenylbutazone N-(2-carboxy-2-hydroxycaproyl)-hydrazobenzene (111) is formed which leads after further hydrolysis to n-carproylhydrazobenzene (IV). This could be further oxidised giving a mixture of cis and trans-azobenzenes ( 2 9 ) . Phenylbutazone forms in aqueous and partially aqueous solutions by a reversible reaction the compound I ( 3 3 ) . The reac- tion rate and the position of equilibrium depend on the solvent, b u t practically not on the pH ( 3 3 ) . The temperature dependence of rate constants is given in Table 6 ( 3 3 ) .

TABLE 6

Temperature Dependence of Rate constants; Phenylbutazone concentration 0.6 M (33) Medium

Temp. 0

C

*20

70.5 80.1 90.8 100.0

56% Tr iethyleneglycol

60.4 70.9 78.9 89.7 100.6

35% N-Methyl-2pyrrolidon + 15% Tr ie thyleneglycol

60.2 70.1 80.7 91.0 100.8

k x 106

k2x10 6

(S-5

(S-5

1.39 3.55 9,94 22.8

0.20 0.58 1.70 4.39

6.9 6.1 5.8 5.2

0.076 0.33 0.81

1.1 0.9 0.9

0.087 0.297 0.750 2.12 5.84 0.061 0.198 0.620 1.55 4.06

-

0.080 0.319 1.20 3.56 10.9

K

k3x10 6

(5-5

-

0.77 9.62 0.51 0.44 0.37

0.030 0.124 0.53 1.98

0.33 0.95 3.83 12.4

7.8 23

505

PHENY LBUTAZONE

The first three compounds are colourless and may be extracted with ether from acidified solution. The coloured product n-caproylhydrazobenzene may be isolated after extraction with chloroform (28). The preparation and separation of decomposition products of Phenylbutazone has been reported in detail by Pawelczyk and Schmid (28,29,33). The UV-spectra of Phenylbutazone and its decomposition products in 0.001% ethanol solution are given in Fig. 10 (28). The degradation patte!:n of Phenylbutazone has been given by Awang as following (38):

(+KOH) C , H r N =N-C6Hs

1x

506

SYED LAIK ALI

The oxidative degradation of phenylbutazone with an acidic potassium permanganate solution and an alkaline hydrogen peroxide solution has also been reported (34). Mass spectral behaviour of decomposition products of phenylbutazone has also been investigated (35,13).Two previously reported but unidentified phenylbutazone degradation products were isolated by chromatography and identified by mass and nmr spectrometry (36). Beckstead (37) has examined the decomposition of phenylbutazone in solid dosage forms. Awang (38) has tested 56 samples of phenylbutazone tablets and 15 samples of phenylbutazone-antacid formulations in the form of capsules and tablets. The degradation of phenylbutazone bulk drug was observed under conditions of accelerated hydrolytic and oxidative decomposition. In presence of magnesium carbonate phenylbutazone is unstable to heat due to chemiesorption ( 3 9 ) . The photodegradation of phenylbutazone in aqueous solution leads to 2-oxocapronic anilide and other compounds. In the methanolic solution n-butyl (methoxy) malonic dianilide is obtained. In aqueous solution in presence of diethylamine the reaction leads to n-butyl-diethylaminomalonic dianilide and other compounds ( 4 0 ) . Phenylbutazone formulations showed no evidence of chemical instability when stored at ambient temperature, 37OC and 37OC with 75% relative humidity. Measurable chemical degradation occured only at 6OoC, with several formulations showing more than 50 % degradation. The extent of degradation can vary among the tablets of the same bottle and between bottles of the same lot (41). Chemical degradation occurs at 37OC also in some phenylbutazone-antacid formulations and was common at 50° and 6OoC (41). Phenylbutazone tablets are also subject to physical instability which is manifested in a decrease of dissolution rate probably due to polymorphism of phenylbutazone (17) or to change of the properties of excipients such as gelatine and or acacia subcoats of sugar-

PHENY LBUTAZONE

507

c o a t e d t a b l e t s (42). The s t a b i l i t y o f p h e n y l b u t a zone i n s o l i d d i s p e r s i o n s c o n t a i n i n g p o l y e t h y l e n e g l y c o l 6 0 0 0 , u r e a a n d p o l y v i n y l p y r r o l i d o n e was i n v e s t i g a t e d by s u b j e c t i n g t h e samples t o acceler a t e d s t o r a g e c o n d i t i o n s . The d e c o m p o s i t i o n o f p h e n y l b u t a z o n e i n c r e a s e d i n p r e s e n c e o f PEG 6000 a n d urea, w h e r e a s p o l y v i n y l p y r r o l i d o n e h a d n o s i g n i f i c a n t e f f e c t on t h e s t a b i l i t y o f t h e d r u g . The e f f e c t o f h u m i d i t y on t h e s t a b i l i t y o f p h e n y l b u t a z o n e f o r m u l a t i o n s was less p r o n o u n c e d t h a n t h e t e m p e r a t u r e (43,44). T h e s t a b i l i t y o f p h e n y l b u t a z o n e is a l s o i m p a i r e d a t 5OoC when f o r m u l a t e d w i t h p o l y s o r b a t e 80 (44). I n s u p p o s i t o r y f o r m u l a t i o n s d e g r a d a t i o n p r o d u c t s o f p h e n y l b u t a z o n e were a l s o i d e n t i f i e d and t h e process was d e s c r i b e d a s m a i n l y o x i d a t i v e (45,46). Dissolution I n r e s p e c t o f d i s s o l u t i o n r a t e of p h e n y l b u t a z o n e PEG 6000 and u r e a had a n a d v e r s e e f f e c t b u t p o l y v i n y l p y r r o l i d o n e was c o n s i d e r e d t o be a s u p e r i o r c a r r i e r (43). The i n i t i a l d i s s o l u t i o n r a t e o f phenylbutazone and d e u t e r a t e d p h e n y l b u t a z o n e ( d - p h e n y l b u t a z o n e ) i n t o a 25 % ( v / v ) e t h a n o l - w a t e r s o l v e n t a t 25OC a n d c o n s t a n t i o n i c s t r e n g t h from a c o n s t a n t - s u r f a c e area p e l l e t was s t u d i e d a s a f u n c t i o n o f a p p a r e n t pH, b u f f e r c o n c e n t r a t i o n a n d s t i r r i n g r a t e ( 4 7 ) . The same a u t h o r ( 4 7 ) h a s p o s t u l a t e d t h a t p h e n y l b u t a z o n e may h a v e a h i n d e r e d d i s s o l u t i o n due to simultaneous r e v e r s i b l e nonins t a n t a n e o u s c h e m i c a l r e a c t i o n which must take place i n t h e a q u e o u s d i f f u s i o n l a y e r i f t h e d i s s o l u t i o n involves rate-determining d i f f u s i o n through t h e aqueous d i f f u s i o n l a y e r . O t h e r a u t h o r s (48) have i n v e s t i g a t e d t h e behaviour o f phenylbutazone i n i t s t r a n s f e r t h r o u g h a d i m e t h y l s i l o x a n e memb r a n e as a f u n c t i o n o f p H v a l u e . 7.

The p r e s e n c e o f s u r f a c t a n t s i n p h e n y l b u t a z o n e f o r mulations caused an increase i n t h e i n i t i a l dissol u t i o n r a t e . The d i s s o l u t i o n d e c r e a s e d a f t e r S t o r a g e (44).

SYED LAIK ALI

508

The dissolution rates of various polymorphic modifications of phenylbutazone has been subject of number of studies (15,16,17,16). Dissolution studies were conducted in a 0.2 m aqueous phosphate buffer of pH 7.5.with the disc method.

a

forms show a slight transition into 6 when and t ey are compressed into discs. The dissolution rate of the p-modification is higher than all other modifications (16). The dissolution rates of various phenylbutazone polymorphs in 0.2 m phosphate buffer,pH 7.5 at 37OC with disc method at 100 rpm are given in Fig 11 (16). In another study (18) the dissolution rates of spray-dried phenylbutazone samples at various drying temperatures were measured at 37OC in the USP dissolution test solutions. The solubility of the samples obtained at 3OoC was 1.5 times higher than that of the sample obtained at 12OoC and the bioavailability of the new form crystals would be expected to compare with the other known forms (18). 8 . Methods of Analysis

Titr imetr Titration'of Phenvlbutazone dissolved in aceton and with an ihdicator bromthymol blue with sodium hydroxide has been adopted as the official method of analysis in european pharmacopea ( 4 9 ) . In USP XX ( 4 ) titration of phenylbutazone is performed with 0.1 N tetrabutyl ammonium hydroxide. Determination of end-point is done potentiometrically using a glass-calomel electrode system. These methods are not specific, as the common decomposition products such as the carboxylic and hydroxy carboxylic acids are also titratable. Bromometric assay with a potentiometric end-point determination has also been performed ( S O ) . Volumetric determinationfof phenylbutazone with chloramin-T (51) and iodine-chlorine solutions are also known (52). The official BP method involves a nonaqueous titration with acetone as solvent (53). A non-aqueous titration in tetramethyl urea a s solvent has also been reported (54). The use of

509

A 1.0-\ I\

F i g . 10. W s p e c t r a o f p h e n l y b u t a z o n e and i t s d e c o m p o s i t i o n p r o d u c t s i n 0.001% e t h a n o l s o l u t i o n s , p h e n l y b u t a z o n e , I , 11, 111.

1

2

3

4

5

T I M E (h)

F i g . 11. Comparison of d i s s o l u t i o n rates of p h e n l y b u t a z o n e polymorphs i n 0.2m p h o s p h a t e b u f f e r p H 7.5 a t 37OC and 100 r p m .

510

SYED LAIK ALI

borhydrides of alkaline metals for the volumetric analysis of phenylbutazone is also known (55). Grav imetry Phenylbutazone has been determined gravimetrically after precipitation with xanthydrol (56,57).

Colorimetric Analysis Determination at 750 nm afte:: reaction with phosphor-molybden-tungstic acid ( 5 8 ) , at 520 nm after reaction of phenylbutazone with iron (111) chloride and 2,2'-dipyridyl have been reported (26). Hydrolytic conversion of phenylbutazone to benzidine and its subsequent diazotization and coupling reaction has also been utilised for its colorimetric determination (59). UV-Spectrophotometry Phenylbutazone can be determined spectrophotometrically after oxidation to azobenzene at 320 nm (60). A uv-spectrophotometric measurement of phenylbutazone in presence of decomposition products, other medicinal agents and interfering dyes involves an acid and base shake-out followed by measurement at 232 and 264 nm (37). Molar absorptivity values of phenylbutazone and its decomposition products in neutral and alkaline solutions are given in Table 7 (37) Chroma tog r aph ic Methods Paper Chromatography Paper of Schleicher and Schull Company as stationary phase and butanol, formic acid and water (12+1+7) or butanol, ammonia 25% and water (84+8+8) as mobile phases have been suggested for the paper chromatographic separation of phenylbutazone (61). Detection was performed with Dragendorff's reagent, diazotized sulfanilic acid and 1% mercury (11) nitrate solutions (61). Thin Layer Chromatography Several systems for the TLC analysis of phenylbutazone and its decomposition products are available. Backstead (37) has reported cyclohexane-chloroform-methanol-acetic acid (60+30+5+5) as solvent system and silica gel GF thin layer plates a s stationary phase to be ideal for resolving and semiquantitatively estimating

TABLE 7

Molar A b s o p t i v i t y Values of Phenylbutazone and i t s Decomposition P r o d u c t s (37) Compound

Neutral Solution

0 . 0 1 N N NaOH

0 . 1 N NaOH ~~

h max

h max

~~

264 nm

nm

t max

Phenyl butazone

24 7

15360

264

20925

20925

Decornp. Prod. I

236

188 10

232

15425

3550

Decomp. Prod. I1

236

17385

232

16400

3835

Decornp. Prod. I11

236

19000

232

16550

3915

Decornp. Prod. I V

236

18455

236

14890

3080

*

nm

L

max

Decomposition P r o d u c t s I , 11, I11 and I V a r e t h e same a s mentioned under 6 ( D e g r a d a t i o n and S t a b i l i t y )

SYED LAIK ALI

512

phenylbutazone and its decomposition products. A solvent system of cyclohexane-methyl ethyl ketonechloroform-acetic acid (39+62+3+10) and silica gel GF 254 thin layer plates were also found to be suitable ( 6 2 ) . Several other solvent systems and silicagel HF and GF254 thin layer plates have been recommended for the TLC of phenylbutazone (61,63,64,65). Silica gel TLC plates previously treated with a 2% solution of sodium hydrogensulfite deters the formation of 4-hydroxyphenylbutazone during the process of development of thin layer plates in solvent system ( 6 6 ) . On-plate oxidation of phenylbutazone was effectively suppressed 1 : 1 mixture of kieselgur and by utilising a silica gel and impregnating the TLC support with Mcllvaine buffer, pH 6.0 (37). Silica gel TLC folies and benzene - acetone (80+20) solvent have been used to separate phenylbutazone from ketophenylbutazone (67)

.

Detection of phenylbutazone and its decomposition products can be achieved by viewing the plates under uv-light. Spraying the TLC plates with chlorine-o-toluidine reagent gives blue to violet coloured spots with a sensitivity well below 1 microgram (37). Folin-ciocalteu reagent gives also coloured spots and is useful in indicating the presence of components which could be overlooked by both uv-light and chlorine-otoluidine reagent (37). Phenylbutazone can also be detected with iron ( 1 1 1 ) chloride in hydrochloric acid, Dragendorff's reagent and dimethylaminobenzaldehyde solution or subjecting the TLC plates to iodine or chlorine vapours ( 6 2 ) . Another spray reagent used was 0 . 5 % potassium dichromate in 20% sulfuric acid (38). A quantitative densitometric determination of decomposition products of phenylbutazone at 232 nm with a Zeiss KM 3 Chromatogramm-Spektrometer has also been reported (66). Gas Chromatography Anachrom ABS/OF-1 qlass column at 200oC isotherm with nitrogen-as carrier gas were used for analysing phenylbutazone through gas chromatography ( 6 2 ) . Perego (68) has given a gas chromatographic method of determination of phenylbutazone in biological fluids in presence of its metabolites

PHENY LBUTAZONE

513

l-phenyl-2-p-hydroxyphenyl-3,5-dioxo-4n-butylpyrazolidine and 1,2-diphenyl-3,5-dioxo-4-(3-hydroxybuty1)-pyrazolidine. A 2.5% SE 30 on Gas Chrom Q, 100-120 mesh glass column was used with nitrogen as carrier gas and flame ionization detector. Quantitation was done by using promazine as an internal standard. Phenylbutazone was extracted from the rat serum or urine after acidifying with 1 N HC1 and extracting with n-heptane (68). Ali (69) has separated phenylbutazone and three of its decomposition products I, I1 and I11 on a 2 m steel column, packed with 3% SE 30 on Varaport 30, 100-120 mesh. Linear temperature programme was run between 19O-25O0C, 6OC/min. Injector and detector temperatures were 230° and 27OoC respectively and nitrogen was used as a carrier gas and flame ionization detector. Mostly decomposition product I was found in phenylbutazone formulations (69). A derivatization procedure for phenylbutazone and other acidic drugs after extraction from plasma and prior to its gas chromatographic determination has been given by Roseboom (70). Phenylbutazone has been chromatographed as its n-butyl ester after treating it with n-butyl iodide in presence of N, N-dimethylacetamide in a methanolic tetramethyl ammonium hydroxide solution. 1.5 m glass columns packed with 3% OV 1, 3% OV 17 and 3% SP 1000, all on chromosorb WHP, 100-120 mesh, were used for the determination of phenylbutazone at 230, 250, and 27OoC column temperatures isotherm respectively. The injection port and detector (FID) temperatures were 3OoC higher than the column temperature. The retention times of phenylbutazone on these three columns were found to be 220, 280 and 202 seconds respectively. Mefenamic acid was used as an internal standard (70). The oxidation of phenylbutazone was studied gas chromatographically on 2 m glass column, packed with 10 % 1,4-butandiolsuccinat on chromosorb W, 80-100 mesh at 13OoC column temperature isotherm (71). It was shown that with alkaline peroxide and acidic permanganate valeric acid is obtained as oxidation product, while with alkaline permanganate butyric acid. 5 % OV 7 on Gas Chrom Q at 15OoC was used for the estimation of intact phenylbutazone and of total degradation impurities in raw material and commercial dosage form with diphenyl phthalate as an internal standard (72). Phenylbutazone has been

514

SYED LAIK ALI

determined in plasma without interference from the metabolites oxyphenbutazone and hydroxyphenylbutazone on 3 % Apiezon L on Chromosorb W-HP, 80-100 mesh at 23OoC isotherm (73). The drug was extracted from plasma after addition of 1 N HC1 with n-heptane. About 9 6 % of phenylbutazone was recovered from plasma through this method. Phenylbutazone and its metabolite oxyphenbutazone have been determined in plasma on a 5 % OV 7 column by flash methylation with trimethylanilinium hydroxide using 1-(o-chloropehnyl)-1-(p-chlorophenyl)-2,2,2- trichloroethane as an internal standard (74). The method described is of sufficient sensitivity to determine plasma levels in humans after a 200 mg dose of phenylbutazone. Oxyphenbutazone and phenylbutazone have been determined in plasma and urine of dogs and horses by GLC on a 3% OV 210 on Gas Chrom Q column at 185OC isotherm (75). A GLC determination of phenylbutazone in human plasma down to 10 ng/ml is reported using 5% OV 17 on Chromosorb WHP column, 80-100 mesh and a 63Ni-electron capture detector (76). A phenylbutazone analog 4-butyl-1,2-bis (p-tolyl)-3,5-pyrazolidine was chosen as an internal standard (76). Fluoranthens as the internal standard and N,O-bis (trimethylsilyl) trifluoroacetamide as the silylating agent have been used to determine phenylbutazone and its metabolites in human or rabbit plasma (77). Another sensitive and specific method for the detection of phenylbutazone in biological samples is its oxidation with permanganate to azobenzene and subsequent gas chromatographic determination on a 10% DC-200 on Gas Chrom Q, 80-100 mesh column with a flame ionization detector ( 7 8 ) . High Per formance !Liquid Chromatography Phenylbutazone and oxyphenbutazone were determined in plasma on a Sil-X-adsorption column using a mobile phase of 0.002% glacial acetic acid and 10-23% tetrahydrofurane in n-hexane with a flow-rate of 1 ml/min at 35OC column temperature and 254 nm detection wavelength (79,SO). 2,4-dinitrophenylhydrazone of benzaldehyde was used as an internal standard. Detection limit for phenylbutazone was found to be 0.2 )Ig/ml. Phenylbutazone and its metabolites oxyphenbutazone and 'y-hydroxyphenylbutazone were determined in plasma and urine through HPLC on

515

PHENY LBUTAZONE

Bondapack C i a column u s i n g a m o b i l e p h a s e o f m e t h a n o l - 0.01M sodium a c e t a t e b u f f e r (pH 4 ) i n a l i n e a r g r a d i e n t (50 t o 1 0 0 % m e t h a n o l a t 5%/min w i t h a f l o w - r a t e o f 2.0 m l / m i n ) a n d a d e t e c t o r wavel e n g t h o f 254 nm ( 8 1 ) . C a l i b r a t i o n c u r v e s f o r a l l t h r e e compounds i n t h e r a n g e s o f 0.5 5 g/ml a n d 5 - 50 g/ml were found t o be l i n e a r . P1 sma a n d u r i n e amples were a c i d i f i e d w i t h H C l , e x t r a c t e d w i t h b e n z e n e - c y c l o h e x a n e (1:l) a n d a f t e r e v a p o r a t i n g t h e s o l v e n t r e s i d u e was t a k e n i n methanol and chromatographed. D e t e c t i o n l i m i t f o r p h e n y l b u t a z o n e a n d i t s m e t a b o l i t e s was 0.05 g/ml ( 8 1 ) . Same a u t h o r s ( 8 2 ) h a v e a l s o r e p o r t e d h e HPLC determination o f phenylbutazone occuring as metabolite o f a n t i - i n f l a m m a t o r y a g e n t s u x i b u t a z o n e i n p l a s m a and u r i n e . c18 Bondapack column was u s e d w i t h methanol-0.5M KH2PO4 a s mob i l e phase i n a l i n e a r g r a d i e n t (from 0 t o 100 % m e t h a n o l a t 8 %/min w i t h a f l o w - r a t e o f 2.0 ml/min) a t 254 nm ( 8 2 ) . M i n u t e q u a n t i t i e s o f p h e n y l b u t a z o n e (50-100 ng/ml) h a v e b e e n d e t e r m i n e d i n plasma, u r i n e , s a l i v a a n d sweat o f h o r s e s t h r o u g h HPLC on a C1a-Bondapack r e v e r s e d - p h a s e column w i t h a m o b i l e phase o f 2 % g l a c i a l acetic i n water-methanol (35+65) a n d a f l o w - r a t e o f 2 ml/min. D e t e c t i o n was a c h i e v e d a t 240 nm ( 8 3 ) . A n o t h e r p r o c e d u r e f o r t h e determination o f phenylbutazone and oxyphenbutazone is b a s e d on a m i c r o p h a s e e x t r a c t i o n from plasma a n d i t s s u b s e q u e n t d e t e r m i n a t i o n t h r o u g h HPLC. A L i c h r o s o r b RP 8 column a n d a m o b i l e p h a s e m e t h a n o l - w a t e r - f o r m i c a c i d (75+250+0.6) were used a t 25OC column t e m p e r a t u r e a n d 1 . 5 ml/min flow-rate. D e t e c t i o n was d o n e a t 238 nm. 0 . 5 g compounds per m l plasma c o u l d b e e a s i l y d e t e r m i l ed r (84).

-

B

P

P

Polarogr aphy The p o l a r o g~-r a p h i c i n a c t i v i t y o f p h e n y l b u t a z o n e a n d o x y p h e n b u t a z o n e a t t h e d r o p p i n g m e r c u r y electrode l e d t o t h e i n t r o d u c t i o n o f a d e r i v a t i z a t i o n proced u r e ( 8 5 ) which i n v o l v e s h y d r o l y s i s o f t h e d r u g i n a m i x t u r e o f acetic and h y d r o c h l o r i c a c i d s , d i a z o t i z a t i o n , c o u p l i n g w i t h 2-naphthol and d i f f e r e n tial-pulse-polarography o f t h e r e s u l t i n g azo-dyes. P h e n y l b u t a z o n e was d e t e r m i n e d i n t h e r a n g e o f lO-7M. P o l a r o g r a p h i c parameters u s e d were f o r c e d drop time: 2 s , p u l s e h e i g h t : 50 mv a n d s c a n - r a t e o f 2 mv/s. Two-electrode o p e r a t i o n was u s e d w i t h a l a r g e s u r f a c e calomel electrode as a n o d e . The

SYED LAIK ALI

516

p o l a r o g r a m was recorded b e t w e e n -0.6 t o -0.8 V ( 8 5 ) . L i n e a r - sweep a n d d i f f e r e n t i a l - p u l s e v o l t a m m e t r i c method for t h e d e t e r m i n a t i o n of phenylbutazone and oxyphenbutazone i n p h a r m a c e u t i c a l d o s a g e forms i s a l s o r e p o r t e d (86). The method i s based on t h e e l e c t r o c h e m i c a l o x i d a t i o n of b o t h d r u g s a t a g l a s s y c a r b o n e l e c t r o d e i n 0.1 M sodium acetate-acetic a c i d i n 98 % e t h a n o l . An i n t e r r u p t e d - s w e e p p r o c e d u r e is g i v e n f o r t h e d e t e r m i n a t i o n of p h e n y l b u t a z o n e i n t h e presence o f oxyphenbutazone (86). 9. Drug Metabolism a n d P h a r m a c o k i n e t i c s S e v e r a l a u t h o r s (.8 7 ,.8 8 . 8 9 , 9 0 1 h a v e d e m o n s t r a t e d t h a t p h e n y l b u t a z o n e u n d e r w e n t aromatic a n d s i d e c h a i n o x i d a t i o n , b u t t h e q u a n t i t i e s i n which t h e i d e n t i f i e d m e t a b o l i t e s were p r e s e n t i n human u r i n e a c c o u n t e d f o r n o t more t h a n a f e w p e r c e n t of t h e dose. T h e s e a m o u n t s i n c r e a s e d o n l y i n s i g n i f i c a n t l y upon c l e a v a g e w i t h p - g l u c u r o n i d a s e . U n a l t e r e d p h e n y l b u t a z o n e d i d n o t c o v e r more t h a n a b o u t 1 % ( 8 7 , 8 8 ) . T h e e l i m i n a t i o n of t h e d r u g from blood i s l a r g e l y d e t e r m i n e d by b i o t r a n s f o r m a t i o n , s i n c e o n l y a v e r y small amount is removed as u n c h a n g e d p h e n y l b u t a z o n e by s t r a i g h t forward r e n a l e x c r e t i o n (87,88). P h e n y l b u t a z o n e u n d e r g o e s b i o t r a n s f o r m a t i o n i n humans t o o x y p h e n b u t a z o n e , - h y d r o x y , p- r - d i h y d r o x y a n d r - o x o d e r i v a t i v e s . T e C-4 g l u c o r o n i d e s of p h e n y l b u t a z o n e a n d v - h y d r o x y p h e n y l b u t a z o n e a r e a l s o known ( 9 1 ) . T h e a b s o r p t i o n f r o m t h e g a s t r o i n t e s t i n a l t r a c t was f o u n d t o be r a p i d a n d complete. A f t e r a d m i n i s t r a t i o n of 1 4 C - l a b e l l e d p h e n y l b u t a z o n e t o a male v o l u n t e e r t h e i n t e g r a t e d c o n c e n t r a t i o n of unchanged p h e n y l b u t a z o n e i n plasma, a s estimated from t h e a r e a u n d e r t h e c o n c e n t r a t i o n c u r v e (AUC b e t w e e n 0 a n d 336 h o u r s was 6 3 % of t h a t of t o t a l 1 4 C - s u b s t a n c e s ( 9 2 ) . T h e c o r r e s p o n d i n g AUCs of t h r e e s p e c i f i c a l l y d e t e r m i n e d metabolites o x y p h e n b u t a z o n e , - h y d r o x y p h e n y l b u t a z o n e a n d p - p i h y d r o x y p h e n y l b u a z o n e were 2 3 %, 2 % a n d 0.5 % r e s p e c t i v e l y ( 9 2 ) . A s i n g l e o r a l dose was s l o w l y e x c r e t e d from t h e o r g a n i s m , s i n c e w i t h i n 21 d a y s o n l y 88 % was r e c o v e r e d , 6 1 % from u r i n e a n d 27 % from faeces ( 9 2 ) . The sum o f s p e c i f i c a l l y measured m e t a b o l i t e s ( o x y p h e n b u t a z o n e , r - h y d r o x y p h e n y l b u t a z o n e , p-r-dihydroxypehnylbutazone) a n d p h e n y l b u t a z o n e i n u r i n e d i d n o t c o v e r more t h a n

1

I!

PHENY LBUTAZONE

517

a b o u t 1 0 %. About 4 0 % a n d 1 2 % o f t o t a l u r i n a r y r a d i o a c t i v i t y was d u e t o C - 4 - g l u c u o r o n i d e s o f p h e n y l b u t a z o n e a n d r - h y d r o x y p h e n y l b u t a z o n e respect i v e l y ( 9 2 ) . Direct g l u c u o r n i d a t i o n of p h e n y l b u t a z o n e i s t h e p r e d o m i n a n t b i o t r a n s f o r m a t i o n process ( 9 2) The time -cour se o f t h e plasma c o n c e n t r a t i o n o f u n a l t e r e d d r u g is c h a r a c t e r i s e d by a n e a r l y maximum o f 36.0 pg/ml a t 3 h o u r s a n d slow d e c a y b e t w e e n 7 a n d 336 h o u r s c o r r e s p o n d i n g t o a n e l i m i n a t i o n h a l f - l i f e o f 88 h o u r s ( 9 2 , 9 3 , 9 4 ) .

.

Ackknowledgement: The a u t h o r t h a n k s D r . K. S c h e i b l i , Ciba-Geigy, Basel, S w i t z e r l a n d f o r p r o v i d i n g some u s e f u l i n f o r m a t i o n .

SYED LAIK ALI

518

References

1) J . R . G e i g y (Basel, S w i t z e r l a n d , German P a t . No 814150 ( 1 9 4 9 ) ; see a l s o R e c h e n b e r g , H.K., Phenylbutazone, G e o r g e Thieme Ver l a g , S t u t t g a r t. 2) J . R . G e i g y ( B a s e l ) , S w i t z e r l a n d , P a t . No. 267222 ( 1 9 5 0 ) . 3) J . R . G e i g y ( B a s e l ) , S w i t z e r l a n d , P a t . No. 3 0 8 1 4 5 ( 1 9 5 5 ) ; C.A. 51, 7 4 2 9 a ( 1 9 5 7 ) . 4) U n i t e d S t a t e Pharmacopeia XX, Page 617 ( 1 9 8 0 ) ; R o c k v i l l e , Md. 2 0 8 5 2 , USA 5) E u r o p a i s c h e s A r z n e i b u c h , Kommentar, P a g e 1073, W i s s e n s c h a f t l i c h e V e r l a g s g e s e l l s c h a f t , S t u t t g a r t (1976). 6) W a l l e n f e l s , K. a n d H. Sund, Drug R e s e a r c h , 9 , 83 ( 1 9 5 9 ) . 7) M a u l d i n g , H.V. a n d M.A. Z o g l i o , J. Pharm. Sic., 60, 309 ( 1 9 7 1 ) . 8) E l - F a t a t r y , H.M., M.M.K. S h a r a f e l - D e e n and M.M. Amer, P h a r m a z i e , 34 1 5 5 ( 1 9 7 9 ) . 9) United S t a t e s Pharmacopeia XX, (1980) R o c k v i l l e , Md. 2 0 8 8 , USA 10) D i b b e r n , H.W., UV and I R s p e c t r a o f some important D r u g s , E d i t i o C o n t o r , A u l e n d o r f (1979). 11) G i r o d , E., R. D e l l r e y a n d F. H a f l i g e r , H e l v . Chim. Acta 40 4 0 8 ( 1 9 5 7 ) . 1 2 ) Ciba-Geigy , P r i v a t e Communication. 13) Locock, R.A., R.E. M o s k a l y k , L.G. C a h t t e n a n d L.M. Lundy, J. Pharm. S c i . , 63 1 8 9 6 ( 1 9 7 4 ) . 1 4 ) U n t e r h a l t , B., Arch. P h a r m a z . , 305 334 ( 1 9 7 2 ) 1 5 ) M a t s u n a g a , J . , W. Nambu a n d T. N a g a i , Chem. Pharm. B u l l . , 24 1 1 6 9 ( 1 9 7 6 ) . Pharm. Acta H e l v . , 5 3 333 ( 1 9 7 8 ) . 1 6 ) M u l l e r , B.W., 1 7 ) I h r a h i m , H.G., F. P i s a n o and A . B r u n o , J. Pharm. S c i . , 66 669 ( 1 9 7 7 ) 1 8 ) M a t s u d a , J . , S . K a w a g u c h i , H. K o b a y a c h i a n d J. N i s h i j o , J. Pharm. P h a r m a c o l . , 32 579 (1980). 1 9 ) Ciba-Geigy, P r i v a t e Communication. 2 0 ) S i n g h , T.P. a n d M. V i j a y a n , C u r r . S c i . , 44 153 (1975). 21) V i j a y a n , M. , C u r r . S c i . , 40 262 ( 1 9 7 1 ) . 2 2 ) S i n g h , T.P. a n d M. V i j a y a n , J. Chem. SOC., P e r k i n 11, 6 9 3 ( 1 9 7 7 ) . 2 3 ) A u t e r h o f f , H. a n d H.P. P a u l i , A r c h . Pharm., 309 538 ( 1 9 7 6 ) .

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B r e n g e l m a n s , J. and G. B r a u n , J. Pharmac. B e l g i q u e , 11 309 ( 1 9 5 5 ) . DAB 7 D D F Akademie V e r l a g , B e r l i n 1 9 8 0 . D e f f n e r , M. a n d A. I s s i d o r i d e s - D e f f n e r , Chim. A n a l y t i q u e , 3 460 ( 1 9 5 8 ) : C.A. 53 1 0 6 6 6 (1959). H e o h l e i n , H., P h a r m a z i e , 21 464 ( 1 9 6 6 ) . P a w e l c z y k , E., R. Wachowiak a n d A. Romanowski, D i s s e r t . Pharm. P h a r m a c o l . , 19 567 ( 1 9 6 7 ) . P a w e l c z y k , E. a n d R. Wachowiak, Dissert. Pharm. P h a r m a c o l , ?o 6 5 3 ( 1 9 6 8 ) . P a w e l c z y k , E. a n d R. Wachowiak, D i s s . Pharm. P h a r m a c o l . , 21 4 9 1 ( 1 9 6 9 ) . P a w e l c z y k , E. a n d R. Wachowiak, D i s s . Pharm. P h a r m a c o l . , 22 4 7 5 ( 1 9 7 0 ) S l i n g s b y , J. a n d D.A. Zuck, Can. J. Pharm. Sci., 2 115 (1972). Schmid, R.W., Helv. Chim. Acta 53 2239 ( 1 9 7 0 ) . Awe, W. a n d H . J . K i e n e r t , Pharm. Acta H e l v . , 38 8 0 5 ( 1 9 6 3 ) . Awang, D.V.C. a n d A. V i n c e n t , J. Pharm. S c i . , 6 5 68 ( 1 9 7 6 ) . M a t s u i , F., D.I. R o b e r t s o n , M.A. P o i r i e r a n d E.G. L o v e r i n g , J. Pharm. S c i . , 69 4 6 9 ( 1 9 8 0 ) . Beckstead, H.D., K.K. K a i s t h a a n d S.J. S m i t h , J. Pharm. S c i . , 57 1 9 5 2 ( 1 9 6 8 ) . Awang, D.V.C., A. V i n c e n t and F. M a t s u i , J. Pharm. S c i . , 62 1 6 7 3 ( 1 9 7 3 ) Monkhouse, D.C. a n d J . L . L a c h , Cand. J. Pharm. S c i . , 7 29 ( 1 9 7 2 ) . R e i s c h , J . , KTG. Weidmann and J. T r i e b e , Arch. Pharm., 310 811 ( 1 9 7 7 ) . M a t s u i , F . , D.L. R o b e r t s o n , P. L a f o n t a i n e , H. K o l a s i n s k i a n d E.G. L o v e r i n g , J. Pharm. S c i . , 67 646 ( 1 9 7 8 ) . B a r r e t t , D. a n d J.J. F e l l , J. Pharm. S c i . , 64 335 ( 1 9 7 5 ) . E l - G a m a l , S.S. , V.F.B. Naggar a n d A.M. Motawi, S c i . Pharm., 49 20 ( 1 9 8 1 ) . N a g g a r , V.F., S . S . E l q a m a l a n d M.A. Shams-Eldeen, S c i . Pharm. $ 3 - 335 ( 1 9 8 0 ) . P a w e l c z y k , E. a n d R. Wachowiak, Acta P o l . Pharm. , 26 4 2 5 ( 1 9 6 9 ) . P a w e l c z y k , E. a n d M. Z a y a c , Acta P o l . Pharm., 27 104 ( 1 9 7 0 ) . S t e l l a , J.V. J. Pharm. S c i . 64 706 ( 1 9 7 5 ) . L o v e r i n g , E.G. and D.B. B l o c k F J . Pharm. S c i . , 63 6 7 1 , 1 3 9 9 ( 1 9 7 4 ) .

-

.

-

.

-

,

-

,

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European P h a r m a c o p o i a , V o l u m e 11, Page 342 (1971), Maisonneuve S.A., F r a n c e . J a n c i k , F., E. Kraus, B. B u d s i n s k y a n d 0. C i n k o v a , Czekoslov.Farmac., 5 105 (1957); C.A. 51 11936 (1957). F e c k o T J . , Acta P o l . Pharm., 21 155 (1964) ; C.A. 62 8942d (1965). G e n g r G o v i c h , A.Y. a n d A.V. S e r d e s h n e v , Aptechn. Delo 2 13 (1960); C.A. 57 1519a

(19621.

B r i t i s h Pharmacopoeia p a g e 364 , (1973), U n i v e r s i t y P r i n t i n g House, Cambridge Walash, M . I . and M. R i z k , I n d i a n J . Pharm.,

39 453

(1977).

B a c h r a t , A.M., Z. BczakovaO L. Knazko and J. B u b e r t , P h a r m a z i e , 32 398 (1977). F r a n c h i , G., Farmaco ( P a v i a ) 12 650 (1957). Mitra, R.K. and G.K. Ray, 1nd.J. Pharm., 25

262 (1963). B r u n o , S. a n d G. L u p o l i , Farmaco ( P a v i a ) , Ed. Sci. 11 462 (1956); C.A. 53 8540 (1959). P u l v e r , R. Schweiz. Med.Wschr , 80 308 (1950). S t a j e r , G., P h a r m a z i e 25 748 (1970). I b e , K . , K.H. Beyer, H T B u r m e i s t e r a n d K.D. Grosser, Arch. T o x i k o l . , 22 349 (1967). E i d e n , F., D e u t s h e A p o t h e k e r Z e i t u n g 107 1522 (1967).

.

G a n s h i r t , H.

and A.

293 925 (1960).

M a l z a c h e r , Arch. Pharm.,

and H.G. T r a c h t , Pharm. Ztg. 108 1365 (1963). Zarnack, J. and S. P f e i f e r , P h a r m a z i e 19 216 (1964). A l i , S.L. a n d Th. S t r i t t m a t t e r , Pharm. Z t g . , 123 720 (1978). Thielemann, H. , Sci. Pharm., 41 336 (1973). P e r e g o , R., E. M a r t i n e l l i a n d P.C. Vanoni, J . Chromatog., 54 280 (1971). A l i , S.L., Pharm. Z t g . , 117 383 (1972). Roeseboom, H. and A. H u l s h o f f , J. Chromatog., 173 65 (1979). S z a b o , A.E.O G. S t a j e r a n d E. V i n k l e r , Arch. Pharm., 307 960 (1974). Watson, J . R . , F. M a t s u i , R.C. Lawrence a n d P.M. McConnell, J. Chromatog., 76 141 (1973). McGilvery, I.J., K.K. Midha, R. B r i e n a n d L. W i l s o n , J. Chromatog., 2 17 (1974). A w e , W.

PHENY LBUTAZONE

52 1

Midha, K . K . , I.J. M c G i l v e r a y a n d C. C h a r e t t e , J. Pharm. S c i . 6 3 1 2 3 4 ( 1 9 7 4 ) . B r u c e , R.B., W . R . Maynard Jr. a n d L.K. D u n n i g , J. Pharm. S c i . , 63 447 ( 1 9 7 4 ) . S i o u f i , A . ? F. C a u d a l a n d F. M a r f i l , J. Pharm. S c i . , 67 2 4 3 ( 1 9 7 8 ) . T a n i m u r a , J . , J. S a i t o h , F. Nakagawa a n d T. S u z u k i , Chem. Pharm. B u l l . , 23 6 5 1 ( 1 9 7 4 ) . Bogan, I . A . , J. Pharm. P h a r m a c . , 2 1 2 5 ( 1 9 7 7 ) . I.J. M c G i l v e r a y a n d R.W. S e a r s , Pound, N . J . , J. Chromatog., 8 9 23 ( 1 9 7 4 ) . Pound, N . J . andR.W. S e a r s , J. Pharm. S c i . , 64 284 ( 1 9 7 5 ) . Marunaka, T., T. S h i b a t a , Y. Minami a n d J. Umeno, J. Chromatog., 183 3 3 1 ( 1 9 8 0 ) . Marunaka, T., T. S h i b a t a , Y. Minami, J. Umeno a n d T. S h i n d o , J. Pharm. S c i . , 69 1 2 5 8 ( 1 9 8 0 ) . A l v i n e r i e , M., J. C h r o m a t o g . , 181 1 3 2 ( 1 9 8 0 ) . S p a h n , H. a n d E. M u t s c h l e r , A r z n e i m . - F o r s c h . , 3 1 495 ( 1 9 8 1 ) . Fogg, A.G. and Y.Z. Ahmed, A n a l y t . Chim. Acta 94 4 5 3 ( 1 9 7 7 ) . Chan, H.K. and A.G. Fogg, A n a l y t . Chim. Acta 109 341 (1979). B u r n s , J.J., R.K. Rose, T. C h e n k i n , A. Goldman, A. S c h u l e r t a n d B.B. B r o d i e , J. Pharmac. Exp. T h e r . , 109 346 ( 1 9 5 3 ) . B u r n s , J.J., R.K. Rose, S. Goodwin, J. R e i c h e n t h a l , E.C. H o r n i n g a n d B.B. Brodie, J. Pharmac. Exp. T h e r . , 111 4 8 1 ( 1 9 5 5 ) . Wagner, J . , H. S t i e r l i n a n d R. P u l v e r , A b s t r a c t s V I I Europ. R h e u m a t o l o g y C o n g r . , B r i g h t o n , England (1971). M c G i l v e r a y , J.J., K.K. Midha and N. Mousseau, P h a r m a c o l o g i s t 1 6 218 ( 1 9 7 4 ) . Midha, K.K., J . P h a r m . S c i . , 63 279 ( 1 9 7 8 ) . D i e t e r l e , W., J . W . F a i g l e , F. F r u h , H. Mory, W. T h e o b a l d , K.O. A l t a n d W . J . Richter, Arzneim.-Forsch., 26 572 ( 1 9 7 6 ) . D a v i e s , D.S. a n d S.S. T h o r g e r i s s o n , Acta P h a r m a c o l . , 2 S u p p l . 3 , 181 ( 1 9 7 1 ) . H v i d b e r g , E.F., P.B. A n d r e a s e n a n d L. Ranek, C l i n . P h a r m a c o l . T h e r . , 15 1 7 1 ( 1 9 7 4 ) .

-

SULFADIAZINE Henry Stober and Wayne DeWitte

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Thermal Gravimetric Analysis 2.8 Microscopy 2.9 Polymorphism 2.10 X-Ray Powder Diffraction 2.1 1 Density and Contact Angle 2.12 Dissociation Constant 2.13 Partition Coefficients 2.14 Solubility 3. Synthesis 4. Inorganic Compounds 4.1 Sodium Salt 4.2 Other Inorganic Compounds 5. Chemical Stability 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Volumetric Methods 6.3 Spectrophotometric Methods 6.4 Column Chromatography 6.5 High Performance Liquid Chromatography 6.6 Gas Chromatography 6.7 Paper Chromatography 6.8 Thin Layer Chromatography 7. Pharmacology

References

Analytical Profilcsof Drug Subrtanccr Volume I I

523

524 524 524 524 524 524 528 528 53 1 53 1 533 533 534 534 536 536 531 531 539 539 539 539 541 54 1 641 54 1 542 542 543 543 543 544 545 546

Copyright 0 1982 by The American Pharmsccuiid Association ISBN 0-12-260811-9

HENRY STOBER AND WAYNE DEWITTE

524

1.

Description 1.1

Name, Formula, Molecular Weight

Generic name Pyrimal; Debenal (1).

-

Sulfadiazine; Sulfapyrimidine;

Nomenclature in Chemical Abstracts:

-

The following nomenclature i s used

4-amino-N-2-pyrimidinyl-benzenesulfonamide -

[68-35-91

Synonyms

- 2 -Pyrimidiny 1sul fani1amide e-Amino-N- (2-pyrimidyl)benzenesulfonamide

N

Structure

0 1 OH 1 ON402S

1.2

Molecular Weight.: 250.27

Appearance? Color, Odor

White or slightly yellow powder. It is odorless or nearly s o , and is stable in air but slowly darkens on exposure to light ( 2 ) . 2.0 Physical Properties

2.1

Infrared Spectrum

The infrared spectrum (Figure 1) was determined for a KBr pellet preparation o f Sulfadiazine USP (2) using a Perkin-Elmer Model 621 IR spectrophotometer. The assignments for important absorption bands are presented in Table I. Assignments were made using general sources ( 3 ) and literature references ( 4 , 5). 2.2

Nuclear Magnetic Resonance (NMR) Spectrum

The NMR spectrum of sulfadiazine was obtained in DMSO-dG containing TMS as internal reference using a PerkinElmer Model R-24B NMR spectrometer (Figure 2). The spectral assignments are given in Table I1 (6).

i 525

526

527

SULFADIAZINE

Table I Infrared Assignments for Sulfadiazine -1

Wavenumber (cm

Assignment

)

3450 3410

N-H symmetric stretching

1650

NH2

1580 1490 1440 1410

Ring skeletal vibrations

1325

SO2 asymmetric stretching

1155

SO2

deformation

symmetric stretching

Table I1 NMR Assignments for Sulfadiazine Multiplicity

No. of P r o t o n s

6.0

Singlet

2

6.7

Doub et

2

7.0

Trip et

7.7

Doublet

8.5

Doublet

11.3

Singlet

Assignment

H2N&

Turczan and Medwick (7) reported similar assignments for sulfadiazine and a classification scheme for the identification of sulfonamides based on their NMR spectra.

HENRY STOBER AND WAYNE DEWITTE

528

The natural abundance 13C magnetic resonance spectrum of sulfadiazine has been reported by Chang and Floss ( 8 ) . Ultraviolet Spectra

2.3

The ultraviolet spectra for sulfadiazine were determined in 0.1M HC1, 0.1M NaOH and USP Simulated Intestinal Fluid, pH 7 . 5 (without enzyme). Solutions were scanned from 350 nm to 200 nm using a Cary 1 4 spectrophotometer ( 9 ) . A summary of the data obtained is presented in Table I11 along with data obtained from the chemical literature. Table I11 Ultraviolet Spectral Values for Sulfadiazine

A 1% 1 cm

Solvent 0.1M- HC1

215 242

548 579

0.1M - NaOH

242 254

821 794

USP Simulated Intestinal Fluid, pH 7 . 5 (without enzyme)

240 254

82 1 794

E thano1

270

844 ( 1 0 )

The ultraviolet spectrum for sulfadiazine in 0.1M NaOH is presented in Figure 3 . 2.4

Mass Spectrum

The mass spectrum of sulfadiazine (Figure 4 ) was obtained with a Kratos Model MS-25 mass spectrometer ( 1 1 ) The mass spectra of sulfapyrimidines have been studied by Cambon and co-workers ( 1 2 ) . For sulfadiazine, preferential fragmentation occurs to eliminate SO, resulting in the fragments observed at m/e = 186 and 1 8 5 . The m/e ass gnments for sulfadiazine are presented in Table IV.

SULFADIAZINE

529

0.8

Solvent: 0.lM NaOH Pathlength: 1.0 cm Concantration: 0.008 mg/rnl

-

0.6

0.4 lu 0

z a

m a

8a

0.2

0.0

'20

240

260

280

300

320

WAVELENGTH (nml

Fig. 3.

UV Spectrum o f S u l f a d i a z i n e

340

530

HENRY STOBER AND WAYNE DEWITTE

?; 20

40

Fig. 4.

Mass Spectrum of S u l f a d i a z i n e

I

1 140

53 1

SULFADIAZINE

Table I V Mass Spectral Assignments for Sulfadiazine ~~~

m/e

Relative Intensity (%)

25 1

0.5

250

0.2

186

90.9

185

100.0

156

5.3

140

3.2

108

22.8

~~

~

Assignment

(M+l)Ot

IP Loss of H from m/e = 186

Q '

U

92

59.4

65

49.6 2.5

t,'

Melting Range

The USP melting range specification for sulfadiazine is 251-254OC (2) u s i n g LISP melting point procedure l a (13). For a sample of Sulfadiazine USP, melting was observed from 253-254OC followed by decomposition (14). 2.6

Differential Thermal Analysis (DTA)

The thermogram for Sulfadiazine USP exhibits a sharp melting endotherm at about 26OOC followed by decomposition (15). A typical thermogram is shown in Figure 5. Sunwoo and Eisen (16) used DTA to determine the melting point and heat of fusion of sulfadiazine recrystallized from acetone. Values of 265.6OC and 7 4 6 4 2 180 cal/mole, respectively, were obtained.

532

HENRY STOBER AND WAYNE DEWITTE

I

-

/

I 26loC

-I-I

ir' 0

3

z

x3 Du Pont 900 DTA N, Atmosphere Heating Rate: 10°C/min. Sample Size: 3 mg

-

A T: 0.5OC/inch

L 50

100

Fig. 5.

L 150

l 200

L 250

DTA Thermogram of S u l f a d i a z i n e

300

533

SULFADIAZINE

2.7

Thermogravimetric Analysis (TGA)

The thermogravimetric behavior of Sulfadiazine USP in a N Z atmosphere was determined with a Perkin-Elmer TGS-1 thermobalance at a scan rate of lO'C/minute (17). For a 3 mg sample a weight l o s s of approximately 0.2% was observed from RT up to 22OoC, followed by a gradually increasing weight l o s s attributed to decomposition at higher temperatures. Cook and Hildebrand (18) have employed TGA for the identification of several sulfa drugs, including sulfadiazine. Scans were conducted at a rate of 5OC/min up to about 800'C. At this temperature no residue of the sulfa drug was left in the sample pan. At lower temperatures SOz is suspected as the major product of pyrolysis. 2.8

Microscopy

Sulfadiazine USP powder is composed of transparent rod-like crystals which exhibit extinction and birefringence under crossed polars. The microscopic crystallographic properties of sulfadiazine reported in the literature have been summarized by Tillson and Eisenberg (19) and are presented in Table V. Table V Microscopic Crystallographic Properties of Sulfadiazine 1

I

Crystal System onoclinic

N 1.596

nclassified 1.615 NOTE:

B

1.675

Ny

Optic Sign 2V

1.830 +

1.663 >1.734

-

I

Ext. Elong. Habit

76' P,i

2

lath shaped

-- P

2

rods

The results reported for the unclassified system are presumed to represent intermediate data which are quite often obtained for some commercial samples and are probably related to hydration.

Sulfadiazine forms characteristic "burrs" of very fine needles when mixed with an acidic solution of gold chloride (20). Treatment of sulfadiazine with a solution of potassium triiodide produces rosettes of blades or needles ( 1 0 ) .

HENRY STOBER AND WAYNE DEWITTE

534

2.9

Polymorphism

An evaluation of polymorphism in sulfonamides has been conducted by Yang and Guillory (21). Sulfadiazine recrystallized from five different solvents did not exhibit polymorphism under the experimental conditions employed. 2.10 X-ray Powder Diffraction The X-ray powder diffraction pattern o f USP sulfadiazine is presented in Table VI (22). Strong lines are observed at 12.8, 21.3, 22.9 and 29.4 degrees 28 for copper Ka radiation. The instrumental and experimental conditions are given below. Instrumental Conditions: G.E. XRD-5: Genera tor : Tube Target: Radiation: Optics :

Goniometer: Detection: Sample Preparation :

Spectrogoniometer 30 kV, 13 mA Cu 0 Cu, Ni Filtered, Ka = 1.542A lo Beam Slit MR Soller Slit 0.2' Detector Slit 3 O Take-Off Angle Scan Rate: 2 degrees 28/minute (5O/inch) SPG-4 Detector Rate Meter, 2000 cps full scale Pulse Height Selection, E = 0.4V, E = 1.5V L U Sample was ground and back-packed into an aluminum sample holder (opening of 3 x 1 x 0 . 2 cm) without sieving. Compound acquires a high static electric charge upon sieving.

SULFADIAZINE

535

Table VI X-ray Powder Diffraction Pattern of Sulfadiazine Major Lines I

28 Degrees$; 7.0 11.7 12.8 14.1 14.6(s) 16.2(s) 16.5 19.0 19.8 20.6(s) 21.3 22.9 24.5 25.9 27.4 28.2 29.4 30.5 33.2 34.6 36.0

12.63 7.56 6.92 6.28 6.07 5.47 5.37 4.67 4.48 4.31 4.17 3.88 3.63 3.44 3.26 3.16 3.04 2.93 2.70 2.59 2.50

10 15 87 26 6 10 14 20 12 5 81 100 7 25 22 18 52 3

3 5 16

;?2@ degrees read to nearest 0 . 1 degrees

interplan planar Distance: d = 2 nh sin

8

“““Relative Intensity in Per Cent Based on Strongest Signal. Signals less than 3% relative intensity were excluded based on 3 (S.D.) o f noise level at 5’ 28. Under the experimental conditions intensities are subject to change sample handling and particle size as a guide for identifying strong (s)

employed, the relative due to variations in and are only included lines.

Shoulder or peak poorly resolved from stronger signals.

HENRY STOBER AND WAYNE DEWITTE

536

2.11 Density and Contact Angle The density and contact angle, 8, of sulfadiazine have been reported by Lerk and co-workers (23). Using sulfadiazine conforming to the specifications of Pharmacopeia Nederlands, values of 1.48 g/cm3 and 28O (powder porosity of 0.168) were obtained.

2.12 Dissociation Constant: Sulfadiazine i s an ampholyte, and in aqueous solutions can exist in the protonated, neutral and anionic forms as illustrated below.

6 "=S=O

&J

- H

\

+ H

0

I

7 @

4

o=s=o

- H

\

+ H

0

Ng I

Salvesen and Schroder-Nielsen (24), using spectrophotometric techniques, reported a pKa value of 2.21 for the anilinium ion associated with sulfadiazine. These authors also stated that the pKa of the pyrimidinium ion

of 2 - s u l f a n i l a m i d o p y r i m i d i n e s is less than zero. Krebs and Speakman (25) used a solubility method to determine the acidity o f the sulfonamide group. A pKa of 6.28 was obtained by these workers, while Willi and Meier (26) obtained a pKa of 6.35 using titrimetry. A summary of the pKa values reported in the literature for sulfadiazine is presented in Table VII.

537

SULFADIAZINE

Table VII Reported pKa Values for Sulfadiazine I

I

Group

CI 2.21

2.00

S02-N-

Referencc

--

24

6.48

Method

Spectrophotometry, 0.5M - NaC1,

T = 24OC

27

Experimental details not stated.

25

Solubility, p = O.lM, - T = 38OC

26

Titrimetry, p = 0.1M - (KCl), T = 2OoC

2.13 Partition Coefficient The partition coefficient o f sulfadiazine was determined for the chloroform/O.lM HC1 and chloroform/O.lM NaOH systems at ambient temperat~res-(s25~C). The partition-coefficient presented in this monograph is defined as K where: P K = [S organic]/[S aqueous] P and [S] is the concentration of sulfadiazine in each phase. System

K

4

CHC13/0.1M- HC1

0.13

CHC13/0.1M- NaOH

0.0005

Sulfadiazine reportedly is extracted from dilute aqueous acid solutions with ethyl ether (10). 2.14 Solubility 2.14.1

Equilibrium Solubility

The following equilibrium solubilities were determined at room temperature (-25OC) and 37OC for a sample of Sulfadiazine USP (28). An equilibration period o f

538

HENRY STOBER AND WAYNE DEWITTE

a b o u t 24 h o u r s was u s e d f o r t h e 3 7 O C c o n d i t i o n ( a g i t a t i o n p r o v i d e d by r o t a t i o n a t 15 rpm i n a c o n s t a n t t e m p e r a t u r e b a t h ) and 72 h o u r s f o r t h e RT c o n d i t i o n ( a g i t a t i o n p r o v i d e d by a w r i s t a c t i o n s h a k e r ) . A n a l y s i s o f t h e c l e a r s o l u t i o n was performed by UV s p e c t r o p h o t o m e t r y . S o l u b i l i t y (mg/ml) RT 37°C -

Solvent - HC1 0.1M

0.61

0.75

Water

0.074

0.10

0.1M p h o s p h a t e b u f f e r (FH 7 . 4 )

0.35

0.67

The s o l u b i l i t y o f s u l f a d i a z i n e i n w a t e r has been r e p o r ted i n t h e l i t e r a t u r e (29) a s approximately 1 g i n 1300 m l ( 0 . 0 8 mg/ml) a t RT and 1 g i n 60 m l ( 1 6 . 7 mg/ml) of b o i l i n g w a t e r . S u l f a d i a z i n e i s a l s o r e p o r t e d a s b e i n g s p a r i n g l y s o l u b l e i n a l c o h o l and a c e t o n e , f r e e l y s o l u b l e i n d i l u t e m i n e r a l a c i d s and s o l u t i o n s o f p o t a s s i u m and sodium hydroxides ( 2 ) . The s o l u b i l i t y o f s u l f a d i a z i n e i n w a t e r and b i o l o g i c a l f l u i d s a t 37°C was r e c e n t l y r e p o r t e d i n t h e Federal Register (30). S o l u b i l i t y (mg/ml) @ 37°C

Solvent Water (pH 5 . 5 ) Serum U r i n e (pH 5 . 5 ) U r i n e (pH 8 . 0 )

0.14 1.60 0.18 4.50

The s o l u b i l i t y of s u l f a d i a z i n e i n s e v e r a l normal a l c o h o l s was r e p o r t e d by Mauger and co-workers ( 3 1 ) . The h i g h e s t s o l u b i l i t y o c c u r r e d i n m e t h a n o l . The H i l d e b r a n d s o l u b i l i t y p a r a m e t e r s o f s u l f a d i a z i n e and o t h e r s e l e c t e d s u l f o n a m i d e s were d e t e r m i n e d by Sunwoo and E i s e n i n s e v e r a l a l c o h o l - w a t e r - g l y c o l s y s t e m s ( 1 6 ) . E l w o r t h y and W o r t h i n g t o n determined t h e s o l u b i l i t y o f s u l f a d i a z i n e i n w a t e r , dimethylformamide and a r a n g e o f m i x t u r e s o f t h e s e s o l v e n t s ( 3 2 ) . 2.14.2

Dissolution Rate

The p o o r s o l u b i l i t y o f s u l f a d r u g s , s u c h a s s u l f a d i a z i n e , i n w a t e r was a f a c t o r i n p r o m p t i n g t h e Food and Drug A d m i n i s t r a t i o n t o e s t a b l i s h b i o e q u i v a l e n c e r e q u i r e -

SULFADIAZINE

539

ments for dosage forms of these compounds. For oral solid dosage forms of sulfadiazine, twelve tablets must be evaluated using the USP Apparatus 11, 50 rpm, 37OC and 0.1M HC1 as the dissolution medium. Specifications of 50% released in 30 minutes and 80% released in 60 minutes were indicated ( 3 0 ) . The intrinsic dissolution rate of sulfadiazine in water was measured by Nogami and co-workers ( 3 3 ) using the rotating disk method. The dissolution of sulfadiazine under these conditions was found to be in accord with the Noyes-Nernst equation concerning transport-controlled processes. In a subsequent publication these authors used the same technique to study the dissolution of sulfadiazine in different aqueous solutions ( 3 4 ) . 3.

Synthesis

The synthesis of sulfadiazine has been described by Roblin ( 3 5 ) . Northey ( 3 6 ) has described the manufacturing procedure used for sulfadiazine. The synthetic reactions consist of condensing 2-aminopyrimidine with p-acetamidobenzenesulfonyl chloride, followed by hydrolysis of the N4-acetyl group with sodium hydroxide. Scheme I depicts the reactions used by Roblin and co-workers. 4.

Inorganic Compounds

4.1

Sodium Salt The sodium salt of sulfadiazine is listed in the

USP

XIX ( 3 7 ) and is employed medically when parenteral therapy

is indicated. Exposure of this compound to humid air results in the gradual absorption of carbon dioxide with the corresponding liberation of sulfadiazine. About 1 gram of sulfadiazine sodium dissolves in 2 ml of water.

4.2

Other Inorganic Compounds

Silver sulfadiazine has found use as an antibacterial agent in the treatment of extensive burns. Spectroscopic data for silver sulfadiazine have been reported in the literature ( 3 8 , 3 9 ) . Bult and Klasen ( 4 0 ) investigated the structure of silver sulfadiazine and indicate that the silver coordinates with both the sulfonamide group and the nitrogens of the 2-aminopyrimidine substituent. A 1 : l complex is formed. Other metallic compounds of sulfadiazine reported in the literature include those of the divalent metals Zn, Cd, Hg, Ni and Mn as well as trivalent Fe ( 4 1 ) .

Scheme I Synthesis of Sulfadiazine

p-Acetamidobenzenesulfonyl chloride

N4 -Acetyl sul fadiaz ine

2-aminopyrimidine

/

hydro 1yze

Sulfadiazine

54 1

SULFADIAZINE

5.

Chemical Stabilitv

Sulfadiazine is stable i n the solid state upon exposure to air, humidity and temperature up to 100°C for two weeks ( 4 2 ) . It darkens upon exposure to light ( 2 ) . Upon pyrolysis sulfadiazine yields 2-aminopyrimidine ( 4 3 ) and sulfur dioxide In solution sulfadiazine undergoes acid-catalyzed (18).

hydrolysis via two pathways. The first pathway yields sulfanilic acid and 2-aminopyrimidine ( 4 3 , 4 4 , 4 5 ) , whereas the second pathway produces sulfanilamide and 2-hydroxypyrimidine ( 4 3 , 4 6 ) . The kinetics of the autoxidation of sulfadiazine i n solution at pH 2 . 0 - 9 . 2 have been reported by Zajac ( 4 7 ) . Methods of Analysis

6.

Elemental Analysis

6.1

The results from an elemental analysis of sulfadiazine are listed below ( 4 8 ) . Elemental Analvsis of Sulfadiazine Element

% Theory

% Found

C

47.99 4.03 22.39

47.80 3.92 22.23

H

N 6.2.

Volumetric Methods

The routine assay of sulfonamides can be accomplished using any of a variety of titrimetric methods. The most widely used assay procedure is titration with sodium nitrite solution to determine the aromatic amine function (49)

-

Several sulfonamides, including sulfadiazine, were determined by titration with KBr03, the end point being indicated by a biamperometric system with platinum-graphite electrodes (50). Agarwal, _ et al. _ (51) have described a spectrophotometric titration procedure for sulfadiazine and other sulfonamides. The drug is dissolved i n a mixture of hydrochloric acid and glacial acetic acid and is then titrated w th a 0.1N bromate-bromide mixture. The endpoint is determined by monitoring the absorbance at 345 nm spectrophotometrical Y.

HENRY STOBER AND WAYNE DEWITTE

542

Greenhow and S p e n c e r ( 5 2 ) have r e p o r t e d a c a t a l y t i c t h e r m o m e t r i c t i t r a t i o n f o r s u l f o n a m i d e s . The d r u g i s d i s s o l v e d i n dimethylformarnide and i s t i t r a t e d w i t h t e t r a n-butylammonium h y d r o x i d e . The e n d p o i n t i s d e t e c t e d by m o n i t o r i n g t h e h e a t e v o l v e d from t h e a l k a l i - c a t a l y s e d a n i o n i c p o l y m e r i z a t i o n o f a c r y l o n i t r i l e , which i s used a s the indicator. 6.3

SDectroDhotometric Methods

The B r a t t o n - M a r s h a l l r e a c t i o n ( 5 3 ) , i n which s u l f a d i a z i n e i s d i a z o t i z e d and t h e n c o u p l e d w i t h N-(1-naphthy1)ethylenediamine dihydrochloride t o give a corored product, i s one o f t h e most commonly used t e s t s f o r s u l f o n a m i d e s . Davis and co-workers ( 5 4 ) r e p o r t a method f o r d e t e r m i n a t i o n of sulfonamides f o l l o w i n g formation of an i n d o p h e n o l dye and measurement o f a b s o r b a n c e a t 725 nm. A method s p e c i f i c f o r s u l f a d i a z i n e i n t h e p r e s e n c e o f o t h e r s u l f a n i l a m i d o p y r i m i d i n e s h a s b e e n r e p o r t e d ( 5 5 ) . The d r u g i s r e a c t e d w i t h 2 - t h i o b a r b i t u r i c a c i d and i s d e t e r m i n e d by measurement of t h e a b s o r b a n c e a t 305 nm.

Sulfadiazine present i n t a b l e t s , solutions f o r i n j e c t i o n , e y e d r o p s , b l o o d o r u r i n e may be d e t e r m i n e d by d i a z o t i z a t i o n , f o l l o w e d by c o u p l i n g w i t h p h l o r o g l u c i n o l u n d e r a c i d c o n d i t i o n s . The r e s u l t a n t y e l l o w c o l o r i s m o n i t o r e d a t 415 nm ( 5 6 ) . 6.4

Column Chromatography

A method f o r t h e s e p a r a t i o n and q u a n t i t a t i o n o f sulfonarnides h a s been r e p o r t e d b y Rader ( 5 7 ) . The p r o c e d u r e i s b a s e d on f o r m a t i o n of i o n p a i r s between t h e s u l f o n a m i d e s and tetrabutylammonium i o n , f o l l o w e d by s e p a r a t i o n on p a r t i t i o n c h r o m a t o g r a p h i c columns. The s e p a r a t e d s u l f o n a m i d e s a r e t h e n d e t e r m i n e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r y .

Another method f o r s e p a r a t i o n of s u l f o n a m i d e s , i n c l u d i n g s u l f a d i a z i n e , i n v o l v e s u s e of t h r e e columns. C e l i t e s u p p o r t m a t e r i a l i s c o a t e d w i t h p h o s p h a t e b u f f e r (pH 1 . 7 o r 7 . 8 ) o r b o r a t e b u f f e r (pH 8.7), t h e sample a p p l i e d and e l u t e d w i t h c h l o r o f o r m . The s e p a r a t e d s u l f o n a m i d e s a r e t h e n d e t e r m i n e d c o l o r i m e t r i c a l l y f o l l o w i n g d e r i v a t i z a t i o n by t h e Bratton-Marshall reaction (58).

SULFADIAZINE

6.5

543

High Performance Liquid Chromatography

Cobb and Hill (59) have reported a normal phase high performance liquid chromatographic separation of sulfonamides. Sulfadiazine was separated from a number of other sulfonamides using a silica column and a mobile phase containing cyclohexane ( 8 5 . 7 % ) , anhydrous ethanol (11.4%) and glacial acetic acid (2.9%). Other separation methods for sulfadiazine in the presence of other sulfonamides and of other antibacterial drugs have been reported, utilizing a wide range of stationary phases. These include reverse phase octadecylsilane packing ( 6 0 ) , reverse phase octylsilane ( 6 1 ) , ion pairing chromatography ( 6 2 ) , cation exchange ( 6 3 ) , and anion exchange ( 6 4 ) . Sulfadiazine has been separated from a number of other sulfonamides by taking advantage of the differing complexation reactions with cadmium(I1) and zinc(I1) ions ( 6 5 ) . The stationary phases were either n-propylethylenediamine bonded to silica, or commercially prepared C18 and C8 columns loaded with 4 - d o d e c y l d i e t h y l e n e t r i a m i n e (in the mobile phase). The effects of metal ion concentration and type are discussed. 6.6

Gas Chromatography

Sulfapyrimidines have been analyzed by gas chromatography following acid hydrolysis to give sulfanilic acid and the respective aminopyrimidines. The aminopyrimidine is then analyzed using a column containing 5% SE-30 and 5% Carbowax 20M on Chromosorb W at a column temperature of 15OOC (66). Sulfadiazine and its principal metabolite, N4acetylsulfadiazine, in plasma or urine, have been analyzed by gas chromatography with electron capture detection following de r vatization with azomethane ( 6 7 ) . 6.7

Paper Chromatography

Sulfadiazine and other sulfonamides have been ana yzed by paper chromatography using several developing solvents and detection methods ( 6 8 ) . The visualization method most suitable for sulfadiazine was reported to be reaction with Ehrlich reagent (p-dimethylaminobenzaldehyde in 1M HC1). The chromatography was performed on Whatman No. 1 filter paper. The R values €or sulfadiazine were 0 . 8 2 , 0 . 7 6 , 0 . 4 5 , f 0 . 0 0 , 0 . 2 4 , and 0.87 in solvent systems A-F, respectively.

HENRY STOBER AND WAYNE DEWITTE

544

The s o l v e n t s y s t e m s used were: A.

Methyl i s o b u t y l k e t o n e - f o r m i c a c i d - w a t e r (10 p a r t s k e t o n e s a t u r a t e d w i t h 1 p a r t 4% formic a c i d ) .

B.

Chloroform-methanol-formic a c i d - w a t e r (10 p a r t s chloroform s a t u r a t e d w i t h a mixture of 1 p a r t methanol and 1 p a r t 4% f o r m i c a c i d ) .

C.

Benzene-methyl e t h y l k e t o n e - f o r m i c a c i d - w a t e r ( a m i x t u r e o f 9 p a r t s b e n z e n e and 1 p a r t k e t o n e s a t u r a t e d w i t h 1 p a r t 2% f o r m i c a c i d ) .

D.

Benzene-formic a c i d - w a t e r (10 p a r t s benzene s a t u r a t e d w i t h 1 p a r t 2% f o r m i c a c i d ) .

E.

Methyl e t h y l k e t o n e - d i e t h y l a m i n e - w a t e r (921:2: 77).

F.

Methyl e t h y l k e t o n e - a c e t o n e - f o r m i c a c i d - w a t e r

(40:2:1:6). 6.8

T h i n Layer Chromatography

S u l f a d i a z i n e c a n be d e t e r m i n e d i n m i x t u r e s o f sulfonamides u s i n g a t h i n l a y e r chromatographic s e p a r a t i o n f o l l o w e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r i c a n a l y s i s (69). The samples a r e s p o t t e d on f l u o r e s c e n t s i l i c a g e l H p l a t e s and developed i n chloroform-methanol (88:12). The s p o t s o f i n t e r e s t a r e s c r a p e d o f f t h e p l a t e a f t e r d e l i n e a t i o n u n d e r UV l i g h t and e x t r a c t e d w i t h 1M NaOH. The a b s o r b a n c e s o f t h e c e n t r i f u g e d e x t r a c t s a r e read w i t h a r e c o r d i n g s p e c t r o p h o t o meter. Walash and Agarwal ( 7 0 ) r e p o r t a s e p a r a t i o n o f s u l f a d i a z i n e from o t h e r s u l f o n a m i d e s . S u l f a d i a z i n e h a s a n Rf o f 0.56 u s i n g s i l i c a g e l p l a t e s d e v e l o p e d w i t h a m i x t u r e o f e t h y l a c e t a t e - m e t h a n o l (9:l). The s p o t c a n b e l o c a t e d u s i n g any o f t h e f o l l o w i n g r e a g e n t s :

(1)

s a t u r a t e d c o p p e r a c e t a t e i n methanol (brown s p o t ) ( 2 ) s a t u r a t e d c o p p e r a c e t a t e i n a c e t o n e (brown) ( 3 ) 5% c o p p e r s u l f a t e i n w a t e r (brown) (4) 2% c e r i c s u l f a t e i n w a t e r w i t h 5 m l o f conc e n t r a t e d H2S04 ( y e l l o w ) .

545

SULFADIAZINE

The chromatographic behavior of a sulfonamides including sulfadiazine has also on strong and weak cation and anion exchange with several matrices using both aqueous and solvent systems (71).

variety of been studied TLC plates nonaqueous

Clarke and Humphreys (72) describe methods for identification of twenty-five sulfonamides using silica gel G plates coated with NaOH, KHSO,, HzO and NaOH and solvent systems 1-4, respectively. The spots are located using N-(1-naphthy1)copper sulfate, p-dimethylaminobenzaldehyde, ethylenediamine-2HC1 or fluorescein. chloroform-methanol (4:l) chloroform-carbon tetrachloride-methanol (7:2:1) ( 3 ) ethylacetate-methanol (9:l) (4) acetone-methanol (4:1)

(1) (2)

The separation of sulfadiazine from two of its known hydrolysis products, sulfanilamide and sulfanilic acid, has been reported using silanized silica gel layers impregnated with triethanolamine dodecylbenzenesulfonate or N-dodecylpyridinium chloride (73). Their behavior and that of other sulfonamides was also studied on layers not impregnated with detergents. Cieri (74) describes a method for the detection of sulfadiazine and other sulfonamides in animal feeds. The sulfonamides are extracted from feed samples with alcohol or acetone and are cleaned up by a Celite column chromatographic technique. The eluate is spotted on an Adsorbosil-1 TLC plate, which is developed in chloroform-methanol (95:5) and then sprayed with an alcoholic solution of p-dimethylaminobenzaldehyde.

7.

Pharmacology

Sulfadiazine is used for the cure of infections originating from susceptible Gram-positive bacteria. It is absorbed readily from the gastrointestinal tract to yield reproducible blood levels. The solubility of sulfadiazine in urine makes it suitable for the treatment o f E. - -coli infections in the urinary tract. The systemic toxicity of sulfadiazine is low, and relatively few side effects are associated with its use ( 7 5 , 76). The chronic toxicity of sulfadiazine has been reported by Schmidt (77).

HENRY STOBER AND WAYNE DEWITTE

546

The metabolism of sulfadiazine in humans involves acetylation ( i n the liver), oxidation and hydrolysis. I n addition to unchanged sulfadiazine, which accounted for over 50% of the products excreted, Uno and Sehine ( 7 8 , 7 9 ) identified the following metabolites in human urine: N4-acetylsulfadiazine, sulfadiazine N4-glucuronide, sulfadiazine sulfonate and sulfanilamide. The analytical method employed by these authors involved separation of the metabolites by paper chromatography followed by elution of each component and analysis using Ehrlichs reagent. The Bratton-Marshall reaction ( 8 0 ) and gas chromatography with electron capture detection ( 6 7 ) have also been employed for the analysis of sulfadiazine in biological fluids. The protein binding of sulfadiazine and other sulfa drugs has been investigated by Hsu and co-workers ( 8 1 ) . They found that binding affinity increased with the number of methyl substituents in the 2-pyrimidine ring. References 1.

Merck Index, 9th ed., Merck and Co., Rahway, N.J., ( 1 9 7 6 ) page 1151.

2. The United States Pharmacopeia XIX, Mack Printing Co., Easton, PA., ( 1 9 7 5 ) page 4 7 6 . 3.

L.J. Bellamy, The Infra-red Spectra of Complex Molecules, Wiley, New York, 1958.

4.

T. Uno, Chem. Pharm. Bull.,

11,7 0 4

(1963).

5. Chem. Abs., 3 9 , 2513e ( 1 9 6 2 ) . Carter, CIBA-GEIGY Corp., Personal Communication

6.

G.

7.

J . Turczan and (1972).

8.

C. Chang and H . Floss, J . Med. Chem.,

9.

D. Madrid, CIBA-GEIGY Corp., Personal Communication.

T. Medwick, J. Pharm. Sci.,

61,

18,505

434

(1975).

541

SULFADIAZINE

References (Continued) 10. E.G.C. Clarke (ed.), Isolation and Identification of Drugs, The Pharmaceutical Press, London, England (1969) page 547. 11.

G. Carter, CIBA-GEIGY Corp., Personal Communication.

12.

A. Cambon, R. Guedj, P. Robert, J. Soyfer and M. Azzaro, Bull. SOC. Chim. Fr., Part 2 , 567 (1970).

13.

The United States Pharmacopeia XIX, Mack Printing Co., Easton, PA., (1975) page 652.

14.

D. Madrid, CIBA-GEIGY Corp., Personal Communication.

15.

J. Quitasol, CIBA-GEIGY Corp., Personal Communication.

16.

60, 238 (1971). C. Sunwoo and H. Eisen, J. Pharm. Sci., -

17.

J. Quitasol, CIBA-GEIGY Corp., Personal Communication.

18.

R.E. Cook and D.A. Hildebrand, Thermochimica Acta,

9,

129 (1974). 19.

A.H. Tillson and W.V. Eisenberg, J. Amer. Pharm. ASSOC., Sci. Ed., 4 3 , 760 (1954).

20.

G.L. Keenan, J. Amer. Pharm. ASSOC., Sci. Ed., 37, 202 (1948).

21.

S . S . Yang and J.K. Guillory, J. Pharm. Sci.,

61,

26

(1972). 22. 23.

J. Quitasol and R. Morris, CIBA-GEIGY Corp., Personal Communication. C.F. Lerk, A.J.M. Schoonen and J.T. Fell, J. Pharm. Sci., 65, 843 (1976).

24.

B. Salvesen and M. Schroder-Nielson, Medd. Norsk. Farm. Selskap., 32, 87 (1971).

25.

H.A. Krebs and J.C. Speakman, Brit. Med. J., (1946).

1,47

HENRY STOBER AND WAYNE DEWITTE

548

References (Continued) 26.

A.V. Willi and W. Meier, Helv. Chim. Acta, 39, 54 (1956).

27.

T.Koizumi, T. Arita and K. Kakemi, Chem. Pharm. Bull., 1 2 , 413 (1964).

-

28.

D. Madrid, CIBA-GEIGY Corp., Personal Communication.

29.

Remington's Pharmaceutical Sciences, 1 4 t h ed., Mack Publishing Co., Easton, PA., (1970) page 1200.

30.

The Federal Register, 44, 60320 (1979) .

31.

J.W. Mauger, A.N. Paruta and R.J. Gerraughty, J. Pharm. Sci.,6 1 , 94 (1972).

32.

P.H. Elworthy and H.E.C. Worthington, J. Pharm. 20, 830 (1968). Pharmacol., -

33.

H. Nogami, T. Nagai and A. Suzuki, Chem. Pharm. Bull., 1 4 , 329 (1966).

34.

H. Nogami, T. Nagai and A. Suzuki, Ibid., - 1 4 , 339 ( 1966) .

35.

R. Roblin, P. Winnek, J . Williams and J . English, J . Amer. Chem. S O C . , 62, 2002 (1940) .

36.

E . H . Northey, Ind. Eng. Chem., 3 5 , 829 ( 1943) .

37.

The United States Pharmacopeia XIX, Mack Printing Co., Easton, PA., (1975) page 477.

38.

B. Sandman, R. Nesbitt and R. Sandman, J. Pharm. Sci.,

63, 948 (1974). 39.

D.S. Cook and M.F. Turner, J . Chem. SOC., Perkin 11, 1021 (1975).

40.

A. Bult and H. Klasen, J . Pharm. Sci.,

41.

Chem. Abs., 9 0 , 214419a, (1979).

42.

D. Madrid, CIBA-GEIGY Corp., Personal Communication.

67,284

( 1978) .

549

SULFADIAZINE R e f e r e n c e s (Continued 1

g4,

43.

H . A u t e r h o f f and U . S c h m i d t , D t s c h . A p o t h . - Z t g . , 1581 ( 1 9 7 4 ) .

44.

V.S. V e n t u r e l l a , J . Pharm. S c i . , 5 7 , 1151 ( 1 9 6 8 ) .

45.

M . Z a j a c , Diss. Pharm. P h a r m a c o l . , 2 2 , 455 ( 1 9 7 0 ) .

46.

M . Z a j a c , P o l . J . Pharmacol. P h a r m . , 2 9 , 689 ( 1 9 7 7 ) .

47.

M . Z a j a c , ~I b i d . , 2 9 , 445 ( 1 9 7 7 ) .

48.

G . R o b e r t s o n , CIBA-GEIGY C o r p . , P e r s o n a l Communication.

49.

The U n i t e d S t a t e s Pharmacopeia X I X , Mack P r i n t i n g C o . , E a s t o n , P A . , (1975) page 6 2 6 .

50.

B . B . P r a s a d , G.D.Khandeliva1 and T . B . S i n g h , Acta P o l . 5,2 0 6 5 8 2 ~( 1 9 7 7 ) .

~Pharm., 3 4 , 177 ( 1 9 7 7 ) ; Chem. A b s t r . ,

51.

S . P . Agarwal, M.I. Walash and M.I. B l a k e , J . Pharm. S c i . , 6 1 , 779 ( 1 9 7 2 ) .

~-

47, 1384

52

E . J . Greenhow and L . E . S p e n c e r , Anal. Chern., (1975).

53

A . C . B r a t t o n and F.K. M a r s h a l l , J . B i o l . Chem., 537 ( 1 9 3 9 ) .

54.

D.R. Davis, A.G. 9 9 , 12 ( 1 9 7 4 ) .

128,

Fogg and D. T h o r b u r n B u r n s , A n a l y s t ,

-

2 , 679

55.

H.W.

56.

R . R . K r i s h n a and C . S . P . S a s t r y , I n d i a n Chem. J . , (1978).

57.

B . R . R a d e r , J . Pharm. S c i . , 6 2 , 1148 ( 1 9 7 3 )

58.

Conroy, J . Assoc. O f f . A g r i c . Chem.,

J . Crommen, J . Pharm. Belg.,

32,

(1954).

13, 27

128 ( 1 9 7 7 ) ; Chem. A b s t r .

8 7 , 90789n ( 1 9 7 7 ) .

59.

P.H. Cobb and G . T . H i l l , J . C h r o m a t o g r . ,

123,444

(1977).

HENRY STOBER AND WAYNE DEWITTE

550

References (Continued) 60.

61.

T . J . Goehl, L . K . Mathur, J . D . Strum, J.M. J a f f e , W.H. P i t l i c k , V . P . Shah, R . I . P o u s t and J . L . C o l a i z z i , J . Pharm. S c i . , 67, 404 ( 1 9 7 8 ) . P . Helboe and M . Thomsen, Arch. Pharm. Chemi, S c i . E d . , Chem. A b s t r . , 87, 90800j (1977).

5, 453 (1977);

62.

S.C. S u , A . V . Hartkopf and B . L . K a r g e r , J . Chromatogr., 119, 523 (1976).

P o e t and H . H . Pu, J . Pharm. S c i . , 62, 809 (1973).

63.

R.B.

64.

T.C. Kram, J . Pharm. S c i . ,

65.

N . H . C . Cooke, R . L . V i a v a t t e n e , R . E k s t e e n , W.S. Wong, G . Davies and B . L . K a r g e r , J . Chromatogr., __ 149, 391 (1978).

66.

J.W. T u r c z a n , J . Pharm. S c i . , 57, 142 (1968).

67.

A . Bye and G . Land, J . Chromatogr., 139, 181 (1977)

68.

L . R e i o , J . Chromatogr., 88, 119 (1974)

69.

U.R.

70.

M.I. Walash and S . P . Agarwal, J . Pharm. S c i . , 61, 277

61,

254 (1972).

C i e r i , J . Chromatogr., 49, 493 (1970).

(1972). 71.

L. L e p r i , P . G . D e s i d e r i and G . T a n t u r l y , J . Chromatogr., 93, 201 (1974).

-

72.

E . G . C . C l a r k e and D . J . Humphreys, J . Pharm. P h a r m a c o l . , 22, 845 (1970).

73.

L . L e p r i , P.G. D e s i d e r i and D . Heimler, J . Chromatogr., 169, 271 (1979).

74.

U . R . C i e r i , J . Assoc. O f f . A n a l . Chem.,

75.

W . Model, H . O .

59,

56 (1976).

S c h i l d and A . Wilson, Applied Pharmac o l o g y , W.B. S a u n d e r s & C o . , P h i l a . (1976) Chapt. 42.

55 1

SULFADIAZINE R ef e r e nc e s ( Co n tin u ed ) 76.

A . Burger ( e d . ) , M ed icin al C h em is tr y, I n t e r s c i e n c e , N e w York, ( 1 9 6 0 ) Chapt. 41.

77.

L . H . Schmidt, J . Pharmacol. & E x p l . T h e r . , 81, 17 ( 1 9 4 4 ) .

78.

T . Uno and Y . S e h i n e , Chem. Pharm. B u l l . , 1 1 , 872 ( 1 9 6 3 ) .

79.

T . Uno and Y . S e h i n e , _ I b i d_. , -1 4 , 687 ( 1 9 6 6 ) .

80.

W. A . R i t s c h e l , G . R i t s c h e l , C . R . Buncher and J . Rotmensch, Drug I n t e l l . C l i n . Pharm., 1 0 , 402 ( 1 9 7 6 )

81.

P . Hsu, J . K . H .

Ma, H.W. Ju n and L . A . L u z z i , J . Pharm.

S c i . , 63, 27 ( 1 9 7 4 ) . ~-

LEVARTERENOL BITARTRATE Terry D.Wilson 556 556 556

1. Foreword 2. Description 2.1 Nomenclature 2.2 Formula 2.3 Molecular Weight 2.4 Structure 2.5 CA Registry Number 3. Physical Properties 3.1 Nuclear Magnetic Resonance 3.2 Mass Spectrum 3.3 Dissociation Constants 3.4 Electron Spin Resonance 4. Stability 4.1 Oxidation 4.2 Dosage Form Stability 5 . Methods of Analysis 5.1 Colorimetric Titration 5.2 UV Spectrophotometric 5.3 Fluorescence 5.4 Radioenzymatic 5.5 lmmunoassay 5.6 Chromatography 6. Metabolism and Pharmacokinetics 7. Acknowledgement

556 556 556 556 556 556 558 560 562 562 562 562 564 564 564 564 565 565 568 577 578 581

8. References

Analytical Profiles of Drug Substances Volume I I

555

Copyright 0 1982 by The American PharrnacculicalAssocialion ISBN 0-12-260811-9

TERRY D.WILSON

556

Foreword L e v a r t e r e n o l b i t a r t r a t e is a s t r o n e a and 6, a d r e n e r g i c a g e n t h a v i n g p e r i p h e r a l v a s o c o n s t r i c t i o n and coronary a r t e r y d i l a t a t i o n Dronerties. It is indicated i n t h e treatment o f a c u t e h y not e ns i ve s t a t e s and cardiac a r r e s t . A d m i n i s t r a t i o n i s done I.V. i n a 5% d e x t r o s e s o l u t i o n i n d i s t i l l e d water o r s a l i n e a t a d o s e o f l - l O a g p e r m i n u t e . 1 - 4 The p r e s e n t s u p p l e m e n t follows the o r i g i n a l Analytical P r o f i l e s review. 5

1.

Description 2 . 1 Nomenclature L e v a r t e r no1 B i t a r t r a t e Levophed8 B i t a r t r a t e ( 2 ) -4- (2-amino-1 - h y d r o x y e t h y l ) - 1 , 2 b e n z e n e d i o l [R- (R*, R*)] -2 ,j-dihydroxybutaned i o a t e (1:l) s a l t , monohydrate 1-Norepinephrine Bitartrate 2 . 2 Formula _. C8H11r403- C4H606 2 . 3 M o l e c u l a r Wei h t h y d r a t e 337 2 a n h y d r i d e 319.27 2.4 S t r u c t u r e 2.

--+

Anhydride r51-40-1]

3.

Physical Properties Nuclear. M a g n e t i c Kesonance The 1 H - n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r u m o f l e v a r t e r e n o l b i t a r t r a t e monohydrate i s shown i n F i g u r e 1. The s p e c t r u m was t a k e n f r o m a 1 0 % s o l u t i o n i n 920 on a V a r i a n HA 1 0 0 s p e c t r o m e t e r w i t h a TMS e x t e r n a l s t a n d a r d . A s s i g n m e n t s o f c h e m i c a l s h i f t s a r e shown i n Table I .

3.1

P

I

I

t I

I/

H nuclear magnetic resonance spectrun of levarterenol Figure 1. ' bitartrate monohydrate.

I I

TERRY D. WILSON

558

Table I I H n . m . r . C h e m i c a l S h i f t Data f o r Levarterenol B it a r t r a t e Chemical S h i f t ppm (TKS) rio. I-! Assignments 7.02-7.3 3 aromatic H 5.11 11 OHx7, IdH,H2C ( e x c h a n g e ) GCH 4.84 2 ClCH 3~ 3 - 3 . 6 2 NCH2 The e a g r e e w i t h p r e v i o u s r e s u l t s o f R e i s c h et who a l s o u s e d Tr-S a s e x t e r n a l s t a n d a r d . It was n o t p o s s i b l e however t o c o n v e r t t h e p r i m a r y a m i n e o f l e v a r t e r e n o l t o a TMS d e r i v a t i v EMDS f o r p u r p o s e s o f a n n . m . r . s p e c t r u m . expressed chemical s h i f t d a t a a s T v a l u e s r e l a t i v e t o a p-dio ane i n t e r n a l standard f o r l e v a r t e r e n o l s o l u t i o n s . $ He was a b l e t o show e v i d e n c e f o r i n t e r m o l e c u l a r s e l f - a s s o c i a t i o n by v a r y i n g c o n c e n t r a t i o n s i n D20 a n d DDriSO s o l v e n t s . Upfield a n d d o w n f i e l d s h i f t s were f o u n d r e s p e c t i v e l y f o r t h e phenyl p r o t o n s with i n c r e a s i n g c o n c e n t r a t i o n s .

a1 '97

3.2

[(lass S p e c t r u m

A mass m e c t r u m o f l e v a r t e r e n o l b a s e i s shown i n F i g u r e 2 t a k e n on a HP 5980A. Xxtens i v e fragmentation of the b i t a r t r a t e s a l t i n the i o n source prevented acquisi.tion of a u s e f u l car; be s p e c t r u m . A weak m o l e c u l a r i o n (M') s e e n a t m/e 169. The b a s e peak a t m/e 139 c a n be a t t r i b u t e d t o M+- CH2NH2. C t Q e r p e a k s s e e n a r e in/e 151, $+ - H 0 ; m/e 111, N; - CHOHCH2NH2 a n d m/e 93, M - H28 - CHOHCH2NH2.

Lass s p e c t r o m e t r y combined w i t h g a s chroma t o g r a n h y (GC-MS) on l e v a r t e r e n o l h a s b e e n r e p o r t e d by s e v e r a l a u t h o r s . T h e s e Tlrocedures i n v o l v e d e r i v i t i z a t i o n a n d m o n i t o r i n g s p e c i f i c m/e Deaks commonly e m p l o y i n g m u l t i p l e i o n d e t e c t o r s ( M I D ) . P e n t a f l u o r o roD'onyl d e r i v a t i v e s have been n r e n a r e d l l t 1 2 ~ f ) 3 s 1 h a n dt h e i o n s o f we753, m o l e c u l a r i o n , 577, M+ - CH2NHCOC2F5; 549, M+ -CH~NHCOC~FS-

533s SPF CTPlJf'l 1C2 RLTENTiOW TIFIE 2 6 139 1 l o @ 3 33 2 5 3 s 65 3 24 ? 140 1. 23 4 LAST 4 152 1 . 1 7 161 I i? 169 1 . 6 4 170 1 7 U I N 273-4 N-150-KN LE'J3P-CD DZRECT IFLET €1 PACE i ie0s . __ -. . SfmPLE

LARCST 4

700.

60..

20..

80.,

60., 48.,

28.

Fig.

2.

Mass spectrum of l e v a r t e r e n o l base.

TERRY D. WILSON

560

CO and 176, 'CHZNHCOC~F~have been monitored. Npentafluorobenzyl-O-trimethylsilyl d e r i v i t i z a t i o n h a s a l s o been c a r r i e d o u t w i t h t h e f o l l o w i n g i o n s r e s u l t i n g : 563, m o l e c u l a r i o n ; 548, M+ CH 355, +M+ - CH2N=CHC6FS; 208, CHz=N+=CHC6F5and 173; C7F5

3.3 D i s s o c i a t i o n C o n s t a n t s No g e n e r a l agreement h a s been reached on t h e v a l u e s of d i s s o c i a t i o n c o n s t a n t s f o r l e v a r t e r e n o l . The r e s u l t s of s e v e r a l r e p o r t s a r e shown i n Table 11. Table I1 Dissociation Constants PKal 8.72

9.3 8 57

PKa2

ph3

9.72

12

4

13

9

10.3

9.73

11.13

reference

16

While pKa3 r e s u l t s from t h e i o n i z a t i o n of t h e second p h e n o l i c group, pKai and pKa as determined by t i t r a t i o n p r o c e d u r e s a r e a s s i g n e 5 t o t h e f i r s t p h e n o l i c and t h e ammonium i o n o r v i c e v e r s a . I t h a s been p o i n t e d o u t 9 t h a t t h e i o n i z a t i o n of t h e s e two g r o u p s does n o t o c c u r i n d e p e n d e n t l y and c a n be t h o u g h t o f as s i m u l t a n e o u s r e a c t i o n s a s p i c t u r e d i n Scheme 1. I i s t h e c a t i o n i c form, I1 i s t h e n e u t r a l , I11 t h e z w i t t e r i o n i c , I V t h e monoanionic and V t h e d i a n i o n i c form. The c o r r e c t s t a t e m e n t s f o r t h e r e l a t i o n between t h e macro- and m i c r o - i o n i z a t i o n c o n s t a n t s are:

w

H

W

Qo 0

X

-

W

5I 0

(u

I

X

0 I

X I

v-0

21 'cu I

0

@ 00 X I

TERRY D. WILSON

562

Ganellin w a s a b l e t o derive a tautomeric e q u i l i b r i u m c o n s t a n t , $, from t h e m i c r o c o n s t a n t s u s i n g t h e r e l a t i o n : K t = a n t i l o g (pkl - p k 2 ) . T h i s gave t h e r a t i o o f t h e z w i t t e r i o n t o t h e unc h a r g e d s c i e s w i t h a v a l u e of 1.8 a t 2 5 O C i n water.

v

3.4

E l e c t r o n Spin Resonance A h i g h r e s o l u t i o n E.S.R. spectrum h a s been o b t a i n e d f o r l e v a r t e r e n o l semiquinone a n i o n r a d i c a l and was e s c r i b e d a s i d e n t i c a l t o t h a t o f e p i n e p h r i n e .I' The p h o t o o x i d a t i o n g i v i n g r i s e t o t h e a n i o n - r a d i c a l i s p i c t u r e d i n Scheme 2.

E.S.R. p a r a m e t e r s f o r t h e a n i o n - r a d i c a l a r e : A(5-H) = 3.58 G, A(7-H) = 3.03 G, A ( 6 - H ) = 0 . 9 6 G, A(3-H) = 0.48 G and g= 2.0044. 4.

Stability Oxidation The o x i d a t i o n o f l e v a r t e r e n o l h a s been t&ays in c lu d shown t o proceed v i a pH-dependa i n g i n t r a m o l e c u l a r c y c l i z a t i a e f5,Ba' and external nucleophilic attack a s s e e n i n Schemes 3a and 3b. These r e a c t i o n s were f o l l o w e d by c y c l i c voltammetry.

4.1

Whereas t h e c y c l i z a t i o n r e a t i o n h a s a n o v e r f b l r a t e c o n s t a n t of 0.066 s e c - E a t pH 6.0 , the nucleophilic r e a c t i o n rate v a r i e s with t h e n u c l e o p h i l e from 0 . 2 2 t o 2100 sec'l f o r t h e amino a c i d s c y s t i n e t o c y s t e i n e r e s p e c t i v e l y a t pH 7.4. 4.2

Dosage Form S t a b i l i t Stabilization of evarterenol i n solution dosage forms h a s been a t t a i n e d by a d d i t i o n of a n t i o x i d a n t s i n v a r i o u s combinations. These i n c l u d e sodium b i s u l f i t e and t h e formula shown i n Table 111. H r e a s c o r b i c a c i d i s t h e p r i n c i p l e a n t i o x i d a n t . 25

__ry

0

M

T

0

0

X

0

0

z

0

0 I

I

0

T

X I

00

T if,

I

X I

00

0

I

w

B'

41

g62 I

A1 t \ /

"h; 0 0

X I

0

-I

0

Y I

cu I 0

0

I

w

U

m u

I

z

11 +

N N

W I-

w

n

TERRY D.WILSON

564

T a b l e I11 L e v a r t e r e n o l S o l u t i o n Dosage Form

% IJo r e p i ne ph r ine

Boric a c i d Ascorbic a c i d Pi- a c e t y 1-P - c y s t e ine Sodium c a r b o n a t e q . s . pH 6 . 0 P u r i f i e d water, q.s.

5.

by W t . 1.00 1.50

0.50 0.50

Methods of Analysig Colorimetric T i t r a t i o n The U.S.P. a s s a y f o r n o r e p i n e p h r i n e b i t a r t r a t e i s c a r r i e d o u t by t i t r a t i n g a s o l u t i o n made by a d d i n g a p p r o x i m a t e l y 500 mg n o r e p i n e p h r i n e b i t a r t r a t e t o 20 m l g l a c i a l a c e t i c a c i d . The t i t r a n t used i s 0 . 1 N p e r c h l o r i c a c i d . 'JJhen t h e c r y s t a l v i o l e t e n d p o i n t i s r e a c h e d , each m l o f 0 . 1 N perchloric acid i s equivalent t o mg O f norepinephrine b i t a r t r a t e anhydride.

5.1

-T-

3*93

5.2

UV S p e c t r o p h o t o m e t r i c

Norepinephrine b i t a r t r a t e i n t h e i n j e c t a b l e dosage form i s determined a c c o r d i n g t o t h e U.S.P. by a s p e c t r a l method. Absorbance o f t h e sample e l u t e d from a s i l i c e o u s e a r t h column i s compared t o t h a t o f t h e s t a n d a r d a t k O ~ g / m l i n 1 i n 350 d i l u t e s u l f u r i c a c i d . The absorbance i s d e t e r mined at>max o f 278 m and a l s o a t two inima: 250 and 300 nm t o c o r r e c t f o r b a s e l i n e . 2 t The p r e s e n c e of sodium s u l f i t e a s a n t i o x i d a n t i n l e v a r t e r e n o l s o l u t i o n dosage forms was shown t o c a u s e i n t e r f e r e n c e i n t h e UV23ssay u n l e s s i t w a s c a r r i e d o u t a t pH 7.0.

5.3

Fluorescence W a n t i t a t i o n o f l e v a r t e r e n o l by f l u o r e s cence measurement p r o v i d e s a s e n s i t i v e " means o f a n a l y s i s . R e a c t i o n s p r o d u c i n g f l u o r e s c e n t Droducts i n c l u d e o x i d a t i o n t o a n ethylenediamine c o n d e n s a t i o n p r o d u c t (EDA) and a t r i h y d r o x y i n d o l e

LEVARTERENOL BITARTRATE

565

p r o d u c t (THI) ,gown i n Schemes 4a and 4 b respectively. e s e methods a r e Zarly d e s c r i p t i o n s of a t t r i b y & e d t o : Weil-Malherbe @ and Anton and Sayre. E l u t i o n from a c a t i o n ex hange r e s i n a t pH 6 i s used w i t h *9 o r without?’ p r i o r aluminum oxide a d s o r p t i o n with h e x a c y a n o f e r r a t e ( I I 1 ) o x i d a t i o n i n t h e f o r m e r method. The THI method was o r i g i n a l l y developed u s i n g t h e same o x i d i z i n g ed t o i n c l u d e an3&odine/ a g e n t b u t h a s bee i o d i d e o x i d a t i o n 91y9’f43 and automation.

5.4

Radioenzymatic S e n s i t i v e methods f o r l e v a r t e r e n o l have been developed u t i l i z i n g a n enzymatic t r a n s f o r m a t i o n of t h e compound t o a r a d i o a c t i v e p r o d u c t which i s measured by l i q u i d s c i n t i l l a t i o n . T y p i c a l s i n g l e isotope d e r i v i t i z a t i o n techniques a r e i l l u s t r a t e d i n Schemes V a and V b . These r e a c t i o n s a r e c a t a l y z e d by t h enzymes catechol-0-methylt r a n s f e r a s e (COMT) ang6ph e ne thanolami ne -Nm e t h y l t r a n s f e r a s e (PNMT) r e s p e c t i v e l y . These g i v e r i s e t o t r i t i a t e d p r o d u c t s when an 3H-labeled coenzyme (S-adenosylmethionine) is p r e s e n t . E a r l y methods were v e r y t e d i o u s and t i e consuming r e q u i r i n g up t o f o u r days p e r sample 3f however they have been s i m p l i f i e d and commercialized. 37 Products of t h e enzymatic r e a c t i o n were s e p a r a t e d by TLC a f t e r e x t r a c t i o n w i t h to1uene:i-amyl a l c o h o l ( 3 : 2 ) , o x i d i z e d t o v a n i l l i n w i t h p e r i o d a t e and counted b li u i d s c i n t i l l a t i o n i n t h e o r i g i n a l methods. 35938,39 L t m t h o d s however e l i m i n a t e d t h e o x i d a t i o n s t e p , go:f1st2 w h i l e a n o t h e r modif i c a t i o n was a double i s o t o p e d e r i v i t i z a t i o n which allowed fg$ concomitant p e r c e n t a g e r e c o v e r y determination.

5.5

Immunoassay

An immunoassay method w a s used by b 5 w a et g t o measure n o r e p i n e p h r i n e . N-maleylnore p i n e p h r i n e was c o n j u g a t e d t o bovine serum a l bumin by a Nannich r e a c t i o n . Following removal of t h e maleyl group, immunization of r a b b i t s gave antiserum s p e c i f i c f o r t h e conjugate.

SCHEME 40

H HO o\ e

N

H

,

;2HI

-2HI

-

+NH2CH2CH2NH2

- NH2CH2CHO

0

H

+NH2CH2CH2NH2

-

-2H

H

-

2H -2H20

H

SCHEME 4 b

FERRICYANIDE PH 6

H

ASCOREATE

NORADRENOC HROME

H

NORADRENOLUT IN

SCHEME 543 COMT

levarterenol

+

3H-nonnetanephrine

3H-rne thy c a d e n o sylrnethionine

SCHEME 5b levarterenol

h - -

f *

3H-epinephrine

3H-methyl-S-adenosylme thionine

TERRY D.WILSON

568

5.6

Chromato ra h y d y e r and P a p e r Chromatography Thin l a y e r and p a p e r chromatographic systems f o r t h e s e p a r a t i o n a n d - d e t e r m i n a t i o n of l e v a r t e r e n o l are l i s t e d i n Table I V . 5

5.62

Gas Chromatography

S p e c i f i c and s e n s i t i v e methods f o r l e v a r t e r e n o l have been developed u s i n g gas chroma t o g r a p h y . Table V i n c l u d e s i n f o r m a t i o n on c o l umn m a t e r i a l , d e r i v i t i z a t i o n , d e t e c t i o n and d e t e c t i o n l i m i t s . Reference 48 c o n t a i n s a review of p r e v i o u s g . c . methods.

5.63 High-Performance L i q u j d Chromatography

Developments i n t h e f i e l d o f h i g h performance l i q u i d chromatography have supplemented former methods f o r l e v a r t e r e n o l a n a l y s i s , e s p e c i a l l y i n b i o l o g i c a l samples where picogram l e v e l s a r e r o u t i n e l y e n c o u n t e r e d . Ease, low c o s t and r a p i d i t y o f o p e r a t i o n i n a d d i t i o n t o s e n s i t i v i t y and s p e c i f i c i t y a r e a f f o r d e d by t h e s e methods. T h i s s e c t i o n i s a r r a n g e d a c c o r d i n g t o d e t e c t i o n methods w i t h a t a b u l a t i o n of column, mobile phase and d e t e c t i o n l i m i t i n f o r m a t i o n i n Table V I . 5.631

Ultraviolet U . V . d e t e c t i o n was used e a r l i n normal phase i o n - p a i r i n g p a r t i t i o n s t u d i e s . 5 6 9 3 d h i l e measurements are u s u a l l y made a t 280 nm, 52 ,53 250 nm was used i n a s t u d y i n which n o r e p i n e p h r i n e enantiomers were s e p a r a t e d u s i n g t e t r a a c e t y l glucopyranosylisothiocyanate and t r i a c e t a r a b i n o 0t h e r pyrano s y l i s o t h i o c y a n a t e d e r i v i t i z a t i o n . s t u d i e s were done o p t i m i z i n g t h e e f f e c t of t h e a c i d , t h e o r g a n i c m o d i f i e r and t h e pH o f t h e mobile phase 55 and d t e r m i n i n g t h e r e v e r s e phase r e t e n t i o n mechanism. 5%

3'

5.632

Fluorescence A comprehensive r e v i e w o f f l u o r e s c e n c e d e t e c t i o n i n catecholamine by H.P.L.C. h a s r e c e n t l y been p u b l i s h e d . includes d r o x y i n d o l e , 52 , escamine, k2 and o - p h t h a l a l d e h y d e p r e and post-column. While T H I d e t e c t i o n was used i n a d i r e c t i n j e c t i o n c o t i n uous f l o w double ion-exchange column s y s t e mz8 ,

'!@'

3rfalysis B39,

,

TABLE N THIN LAYER AND PAPER CHROMATOGRAPHY OF LEVARTERENOL R ef e r e n ce D e t e c t i on Medium Solvent

Avicel

1st d i m e n s i o n : ( m i c r o c r y s t a l - 1 - b u t a n o l :MeOH: l i n e c e l l u 1 o s e ) l N formic a c i d (60 :20 :20) 2nd d i m e n s i o n : CHC13 :MeOH : 1 N NaOH ( 6 0 : 3 5 : 5 )

C e l l u l o s e MN 300Ecteola

( a n i o n ex-

changer )

Cellulose phosphate paper

Diazotized p-nitroaniline

43

aqueous e t -

44

1 - b u t a n o l :EtOH: O.5N HOAc (38: 8.5 :20)

hylened i a m i n e (1:4)

1 - b u t a n o l :pyri d i n e :H20

0.5% p o t a s sium f e r r i -

( 1 4 :3:30)

cyani de

1 - b u t a n o l :EtOH (95%):H20 (1:1:

chloride

1)

ferric

45

TABLE V

Column

GAS L I Q U I D CHROKATOGRAPHIC ANALYSIS o f LEVARTEKNOL Detector De r i v i t i z a t i on Detection L i m i t s Reference

1%SE-30 1%O V - 1 7

electron capture

pentafluorobenzylimine trimet h y l s i l Yl

5% SE-30

electron capture flame i o n ization

Dentafluoro benzaldehyde b i s- t rime t h y l sil y l a c et a m i d e

3% O V - 1 7

flame i o n iz a t i o n

trimethylsilylimidazole

7'% D C - 1 1

d u a l flame ionization

3% ov-1

electron capture

100 Pg

1.4x1 0-'6rnol/sec

15

46

2.1x10-13mol/sec 2 5 ng

47

b i s- t rime t h y l silylacetamide trif l u o r o a c e t amide

0 . 1 Pg

48

2,6-dinitro-

0.5 Pg

49

4- t r i f l u o r o -

me t h y l b e n z e n e s u l f o n i c acid

TAELE V I

HIGH-PERFORb ANCE L I Q U I D CHRGh ATOGRAPHIC SYSTEP S Ultraviolet Detection

1.

Column S i l i c a gel

LiChrosorb S I 1 0 0 VI

2 AA

Bondanak c18 Nucleosil C 1 8

L o b i l e Phase

for

LEVARTERENOL

Detect i o n Limit

Reference

Bu0H:Hexane ( 1 : l ) Et0Ac:Hexane (9:l) BuOH:MeC12(2:3) ( 0 . 1 M HCPQ4 s t a t io n a r y ) Bu0H:NeCl (4:6) ( 0 . 2 5 M H&O4 s t a t io n a r y )

0.1AAg

50

1 . 0 pmole ml

51

0 . 1 7 ~HOAC PH 2.6 0.67M p h o s p h o r i c a c i d

5 ng

52

40

~g

53

ml O.D.S.

S p h e r i s o r b 55 S i l i c a Porasil- 6 0 S ~ilica LiChrosorp RP-8

1 0 mM p h o s p h a t e b u f f e r pH 2 . 8 : MeOH aqueous p e r c h l o r i c , acetic, chloroacetic, dichloroacetic acids 0.1M H3PO4 pH 3 . 0 , N a o c t y l s u l f a t e : 1.15 $ pentanol

-

54

-

55

-

56

TABLE V I ( c o n t i n u e d )

HIGH-PERFORKANCE L I Q U I D CHROKATOGRAPHIC SYSTEGS 2. Fluorescence D e t e c t i o n Column Zipax SCX Zorbax ODS

CII 4

E o b i l e Phase

for

LEVARTERENOL

Detection L i m i t

0.08M NaH2Po4 pH 4.3 0.1K RaH2POq pH 3.15

Reference

20 Pg

58

1 Pg

Zipax Zipax

SCX

59 60

Zipax

SCX

61

SCX

0.15M NaH2P04

hl

H i t a c h i 3011 g e l 301 0-OH

Me0H:Tris H C 1 pH 8 (7:3) MeOH:O.lSM b o r a t e pH 8

0 . 1 nmol

62

(7:3) TSK g e l 160 TSK-LS 160

Me0H:Tris H C 1 0.5M pH 8

100

ng

63 64

(90:lO)

50 ‘ y g

1 Pg 7.5 ng

65

Bondapak SCX

O . l 5 K NaH2PO4 pH 4.37 C i t r i c acid/HOAc/NaOAc/ NaOH pH 2.8

Zipax SCXCDR-20 a n i o n exchange

0.07M NaH2PO4

10 Pg

68

Zipax

SCX

T r i s HCl:CH3CN pH 8.4

67

TABLE VI (continued) H 1GH-PERFOXti.ANCE LIQUID CHROI..ATOGRAPHIC SYSTEP?S 2.

3. m W 4

Fluorescence Detection Column Nobile Phase TSK-LS 160 CH CN:O.OSM imidazo?e HC1 gH 7.3: Na2EmA 0 mg/L Electrochemical Detection Zipax SCX Vydac SCX Carasil/CX

c18

Nucleosil SA Zipax SCXVydac SCX

0 . 1 ~~ ~ 1 0 4 0.01M H2SO4./ 0.04 M NaS04 acetate/citrate buffer pH 5.2 0.1M citric acid: O.lN! NazHPOh (3:2) 0 . 3 mM Na octane s u l fate citrate/acetate buffer pH 5.2 citrate/acetate buffer pH 5.1

for LEVARTERENOL

Detection Limit 25 fmol

Reference 69

74 75 76

77

78 79

TABLE V I

(continued)

HIGH- PERF0 RIk.ANCE L I Q U I D E O G i ATOG RAPH I C SYSTEMS

3.

Electrochemical Detection Kobile Phase Column 0.1M HNO3 pH 3.0 A Bondapak C18 octane s u l f a t e M Bondapak

c18

ABondapak c18 Vydac

-A.(

SCX

Bondapak Phenyl Bondapak C18 Uhatman SCX

N u c l e o s i l SA

for

LEVARTERENOL

D e t e c t i on -Limit

-

Reference 80

81

citrate/phosphate b u f f e r : r eOH (85:15) pH 3.3 Na o c t a n e sulf o n a t e 2 . 5 ~10-3M EDTA O.lMNaH2P04 0.lM EDTA citrate/acetate buffer

82 83

N a H PO4 b u f f e r pH 5.5 MeOfirH20 pH 4.2 0 . 0 0 2 M PIC B7 o r l38

84

0.008M c i t r i c a c i d /

85

O.Ol3M a c e t a t e pH 5.2

0.01 mM EDTA c it r a t e / a c e t a t e buf f e r pH 5.2

0.25 ~ O I / L

86

TABLE V I ( c o n t i n u e d ) H IGH-PERFORMA NCE L I Q U I D CHRONATOG RAPHIC SYSTEDS

3.

for

LEVARTERENOL

Electrochemical Detection Column

N o b i l e Phase

Detection L i m i t

Re f e r e n c e

MBondapak c18 U l t r a s p h e r e ODS S p h e r i s o r b ODS Biophase ODS

0.lK p h o s p h a t e b u f f e r pH 2.8 N a o c t a n e s u l f a t e 9mg/~ o r 0 . 1 5 ~ c h l o r o a c e t i c a c i d pH 3.l/l mN;: EDTA/Na o c t a n e s u l f a t e 25 mg/L

25 Pg

87

4Bondapak C 1 8

0.1M c i t r a t e : O . l N phosphate b u f f e r ( 3 0 0 : 160) N a octane s u l f a t e 0 . 0 1 g/L

1

88

VI 4

v,

M

Bondapak c18

N a O A c pH 4.8/ 7% CH C N , N a h e p t a n e su f o n a t e

4

U l t r a s p h e r e ODS

citrate/phosphate buff e r pH 4.85/ 14% MeOH 3 mN: N a o c t a n e s u l f a t e

A4

g/L

TABLE V I ( c o n t i n u e d ) H I GH -PERF0 RMA NC E --

3.

L I QU I D CHXOKATOGRA PH I C S Y STEP!,S

for

LEVARTERENOL

Electrochemical_ D e t e c t i o n Column Kobile Phase Uetection L i m i t 0 . 0 3 pmol 0 . 1 ~~ ~ 1 0 4 Zipax SCX B r i tton-Robinson Yanapak ODs40 ng A1203 b u f f e r pH 1.8/0.5 mM N a h e p t a n e s u l f o n a t e and T r i s pH 8.8/0.25% EDTA N u c l e o s i l C18

c i t r a t e/phosphat e buf f e r pH 6.5fiteOH

N u c l e o s i l c18

NeOH:citrate/phosphate b u f f e r pH 5.2 (1:20)

5 0 0 Pg

1 ng/mL

Reference

96 97

98

99

LEVARTERENOLBITARTRATE

577

a chemiluminescent f l u o r e s c e n c e d e t e c t o r h a s been u t i l i z e d by r e a c t i n g bis(2,4,6-trichlorophenyl) ox a la te with fluorescamine l a b e l e d l e v a r t e r e n o l . T h i s method gave a 20-fold i n c r e a s e i n s e n g b t i v i t y over conventional fluorescamine l a b e l i n g . 5.633 E l e c t r o c h e m i c a l The f i e l d of e l e c t r o c h e m i c a l d e t e c t i o n of o x i d i z a b l e compounds s e p a r a t e d by H.P.L.C. h a s s e e n a r a p i d expansion i n r e c e n t y e a r s f o l l o w i n g e a r l y work of P . T . K i s s i n g e r et a -l . Recent reviews o f e l e c t r o c h e m i c a l d e t e c t i o n o f catecholamines i n c l u d e t h e following:70-73. The e l e c t r o c h e m i c a l d e t e c t o r i n g e n e r a l c o n s i s t s of a t h i n - l a y e r o r w a l l - j e t s e c t i o n connected t o a r e s e r v o i r c o n t a i n i n g t h e r e f e r e n c e ( Ag/ AgCl o r S.C.E.) electrode plus an auxiliary e l e c t r o d e , The working e l e c t r o d e o r anode p l a c e d i n t h e t h i n - l a y e r ( 0,002-0.015 i n . ) s e c t i o n c o n s i s t s of a w e l l f i l l e d with c a r b o n p a s t e , a g l a s s y c a r b o n e l e c t r o d e o r a gold/mercury e l e c t r o d e . A number o f s t u d i e s have been cond u c t e d u s i n g each of t h e s e e l e c t r o d e s f o r t h e d e t e c t i o n of l e v a r t e r e n o l . R e f e r e n c e s u s i n g carbon p a s t e e l e c t r o d e s include3 74-90, w h i l e g l a s s y c a r b o n e l e c t r o d e s were sed i n 9195. I n a d d i t i o n d u a l c a r b o n p a s t e $ and d u a l g l a s s y carbon e l e c t r o d e s 97 have been u s e d . A r e c e n t development i s t h e u s e of a r o t a t i n g d i s c e l e c t r o d e w i t h which it i s p o s s i b l e t o d e c r e a s e the d i f f u s i o n l a y e r thickness, t h u s i n c r e a s i g t h e 99 e f f e c t i v e d e t e c t i o n volume and s e n s i t i v i t y . $9

5.634

Radioenzymatic The p r o d u c t s o f t h e r a d i o enzymated r e a c t i o n of l e v a r t e r e n o l i n t h e system d e s c r i b e d i n 5.4 above u s i n g catechol-0-methylt r a n s f e r a s e have been s e p a r a t e d by H.P.L.C. with ormal phase 51 and r e v e r s e d - p h a s e systems. 53,1& Following f r a c t i o n c o l l e c t i o n , samples were counted by l i q u i d s c i n t i l l a t i o n . Metabolism and Pharmacokinetics The metabolism o f l e v a r t e r e n o l h a s been o u t l i n e d i n t h e o r i g i n a l monograph. 5 L i t t l e was known of t h e pharmacokinetics of t h i s d r u g howe v e r u n t i l t h e v e r y s e n s i t i v e methods p r e s e n t l y a v a i l a b l e were developed. One d i f f i c u l t y encount-

6.

TERRY D.WILSON

578

e r e d i n t h e s e measurements i s a d i s t i n c t i o n which must be made between endogenous n o r e p i n e p h r i n e and i t s m e t a b o l i t e s and t h e exogenous d r u g . T h i s i s normally done by r a d i o a c t i v e l a b e l i n g . E a r l y human s t u d i e s were done w i t h t h e a d m i n i s t r a t i o n o f 3H-d,l-norepinephrine and t h e c o l l e c t i o n o f u r i n a r y e x c r e t i o n data. A t r i e x p o n e n t i a l e x p r e s s i o n with a t h r e e compartment model e x p l a i n e d t h e data when 0.02.ug/kg was i n f u s e d i n one h o u r . The h a l f - l i v e s o b t a i n e d from t h e t h r e e s e c t i o n s of t h e u r i n a r y s p e c i f i c a c t i v i t y / t i m e c u r v e were: 1.19 h r . , 5 . 2 2 h r . , and 23.43 h r . The r a t e o f endogenous n o r e p i n e p h r i n e u t p u t was found t o be A s i x compartment 0.022kc g/mg c r e a t i n i n e . model w i t h a b i e x p o n e n t i a l e q u a t i o n was used t o e x p l a i n g t h e r u r i n a r y e x c r e t i o n data. lC2 Here 8 . 3 ~10- , u g / k g min d , l - n o r e p i n e p h r i n e was a d m i n i s t e r e d f o r 48 h o u r s . An a v e r a g e o f 2 7 a g o f f r e e Ilor-epi appeared i n t h e u r i n e w i t h 5 8 1 4 g~ of m e t a b o l i t e s i n 24 h o u r s . I n t h e s e two s t u d i e s f l u o r e s c e n c e and l i q u i d s c i n t i l l a t i o n measurements were used.

lot

Nore r e c e n t l y 3 H - l e v a r t e r e n o l i t s e l f h a s been a d m i n i s t e r e d by i n f u s i o n and plasma k i n e t i c data o b t a i n e d u s i n g t h e s e n s i t i v e r a d i o e n z y m a t i c methods. R e s u l t s o b t a i n e d a r e shown i n T a b l e VII. L e v a r t e r e n o l l e v e l s have been d e t e r m i n e d a l o n g with m e t a b o l i t e s i n human u r i n e , plasma and CSF u s i n g s e v e r a l o f t h e methods d i s c u s s e d above. These a r e summarized i n Table V I I I .

7.

Acknowledgement The a u t h o r wishes t o e x p r e s s h i s t h a n k s t o Mr. A l l a n G. Hlavac o f S t e r l i n g - W i n t h r o p Research I n s t i t u t e f o r c o n t r i b u t i o n s t o t h e Nuclear Magneti c Resonance and Mass Spectrum s e c t i o n s .

TABLE VII LEVARTERENOL PHARNAC 0 K I N E T I C S

ZERO-ORDER INFUSION PLASMA DATA --

Radioenzyme Method

C OMT

Infusion Rate

Infusion Duration

Average t 1/2 m in

-

0.002 g/min

90 min

0.01-

1 0 min

1.45

10 h r

2.09

60 min

2.4

Endogenous Total Clearance Production L/mi n Rate 2.8

AA

PNNT

4-5

0.075

0.06~g/ kg min

C ONT

0.1 g/min

AA

5

0

9

54 %/

m 2 min

0.83-0 - 8 5

Average Reference Plasma Level pg/mL 240

103

190

104

228

105

A g/min

3.07

0 . 7 fig/

min

TABLE V I I I REFERENCES

Nethod

for LEVARTERENOL Urine

Plasma

33

33 48 36, 40, 41

f1uore scence

48

GLC

A N A L Y S I S in HLJhAN F L U I D S

radio-enzymatic

CSF 40

HPLC REFERENCES

ETe thod

GC -MS

uv HPLC

for

LEVARTERENOL M E T A B O L I T E A N A L Y S I S

Urine

106, 107, 108 lo9

Plasma

HUMAN F L U I D S

CSF

107

111

LEVARTERENOL BITARTRATE

8.

1. 2.

3. 4.

5. 6. 7.

58 1

References P h y s i c i a n ' s Desk Reference, C h a r l e s E. Baker, J r . , Kedical Economics C o . , O r a d e l l , N . J . , p .

691 (1981).

s-

O s o l , A . , e d . , Remington's Pharmaceutical ence, 1 6 t h e d i t i o n , Back P u b l i s h i n g Co., Easton, P a , , p. 826 (1980). Goodman, L.S. and Gilman, A , , e d , , The Pharmacolo i c a l Basis of Thera e u t i c s , 5 t h e d i t ~ T k E ' i T l ~ Ye w h . 491 (1975). Foye, d.O., e d . , P r i n c i p l e s of h e d i c i n a l Chemi s t r y , 2 n d e d i t i o n , Lea and F e b i g e r , P h i l a d e l p h i a , P a . , p.377 (1981). Schwender, C . F . , A n a l y t i c a l P r o f i l e s of Drug Substances, Vol. 1, Academic P r e s s , New York, N . Y . , p. 149 (1972). Reisch, J . , A l f e s , H . , and lkollmann, H . , Anal. Chem., 238, 29 (1968). M i w a , A , , Yoshicka, M., S h i r a h a t a , A . , and Tamura, Z., Chem. Pharm. Bull., 25, 1904

z.

(1977)

8. F o r r e s t , J . , Heacock, R . , and F o r r e s t , T . , J. Pharm. Pharmac., 22, 512 (1970). 9. E r a n o t , J . , FEBS L e t t e r s , 67, 271 (1976). 10. Loon, B., P a l , V . , and Waynert, E . , Molec. -' PCol

, 19, 44 (1981).

12.

Koslow, S., C a t t a b e n i , F . , and C o s t a , E., S c i e n c e , 176, 177 (1972). Koslow, S., F r o n t i e r s in Catecholamine Research, Usdin, E.and Snyder, S . , e d . , Pergamon P r e s s , New York, N . Y . , p . 1085

13. 14.

Koslow, S., Racagni, G . and Costa, E . , Neuropharm., 13,-1123 (1974). Hashimoto, Y., and F i y a z a k i , H . , 2. Chromat.

11

I

(1973)

168, 59 (1979)-

15. Lhuguenot, J . C . , and Maume, B., J . Chromat. S c i . , 12, 411 (1974). 16. m a n , K., Davis, J . , Colburn, R . , and J a r k e ,

F., J . Neurochem., 19, 1099 (1972). G a n e l l i n , C . , J . E. Chem., 20, 579 (1977). 18. Yoshioka, b.., K i r i n o , Y . , Tamura, Z . , and Kwan, T . , Chem. ma&. - B u l l . , 25, 7.5 (1977). 19. Hawley, M., Tatawawadi, S . , P i e k a r s k i , S . , and Adams, R . , J . &. Chem. SOC., 89, 447 (1967). 20. Tse, D., IkcCreery, R . , and Adams, R . , J. Chem., 19, 37 (1976).

17*

E.

TERRY D.WILSON

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Chavdarian, C . , Karashima, D . , and C a s t a g - noli, N . , J . P’ed. Chem., 2 1 , 548 ( 1 9 7 8 ) . U.S. P a t e n t HechT, G T a n d Bigelow, N . , number 3 , 8 0 8 , 3 1 7 , The U.S. Pharmacopeia,20th e d i t i o n , U.S.P. Convention, H o c k v i l l e , Did., p. 554 (1980). i b i d , p. 5 5 5 . J u h a s z , F. and P a a l , T . Gyogyszereszet, 2 5 , 2 1 1 ( 1 9 8 1 ) . RINSDOC 41673N. T i e t z , N . , e d i t o r , Fundamentals of C l i n i c a l d . B . Zaunders. P h i l a Chemistry, 2nd ed delDhia. P a . , D. 809 (1976). Weil-Palherbe ,-H, and Bone, A . , Biochem. J.,

21.

22. 23.

-

24. 25 26.

..

27.

51, 311 ( 1 9 5 2 ) .

28. 29 30

9

31 32

33

34

9

35 36

37 * 38 *

39

0

40.

41. 42.

43 *

Anton, A . , and S a y e r , D . , 2. Pharm. Exp. T h e r . 138, 360 ( 1 9 6 2 ) . &ki,-T., 2. Chromat., 155, 415 ( 1 9 7 8 ) . Ogasahara, S . , Kandai, T . , Yamatodani, A . , Watanabe, T . , Wada, H . , and S e k i , T . , J . Chromat., 1 8 0 , 119 (1979). O’Hanlon, J . , Campuzano, H . , and Horvath, S . , Anal. Biochem., 34, 568 ( 1 9 7 0 ) . T e t c a l f , G . , Anal. Biochem., 57, 316 (1974). Campuzane, H . , Wilkerson, J . , and Horvath, S . , Anal. Biochem., 64, 578 (1975). Westerink, B . , and K o r f , J . , J. Neurochem., 29, 697 (1977). Coyle, J . , and Henry, D . , J . Neurochem., 21, 61 ( 1 9 7 3 ) . Henry, D . , Starman, B., Johnson, D . , and Williams, R . , L i f e -” Sci 16, 375 (1975). Up john D i a g n o s t i c s , C a t - A - K i t TM P a s s o n , P, and P z u l e r , J . , Anal. Biochem., 51, 618 ( 1 9 7 3 ) Engelman, K . , and Portnoy, B . , C i r c . Kes., 2 6 , 53 ( 1 9 7 0 ) . P e u l e r , J . , and Johnson, G . , L i f e S c i . , 21, 625 (1977). Bauce, L . , T h o r n h i l l , J . , Cooper, K . , and Veale, vJ., L i f e S c”i 2 7 , 1921 ( 1 9 8 0 ) . S a a r , h . , Bachmann, A . , and Gordon, R . , C l i n . Chem., 2 7 , 626 (1981). E G i n g , R . , and C l a r k , d . , 2. Chromat., 5 2 , 305 ( 1 9 7 0 ) .

-

583

LEVARTERENOLBITARTRATE

..

45 46.

9

47

9

48.

Anal. Biochem. , 51, 42 ( 1 9 7 3 ) . Levin, J a n d H o r n i n g , E. , B i o c h , B i o p h . bio f f a t ,A A c t a , 222 248 (1970):~ Rlaruyama , Y . , a n d Takemori , A , , Anal. chem., 4 9 240 (1972). J . Chromat., L o v e l a d y , H . , a n d F o s t e r , L .’ * 108, 43 ( 975) D o s h i , P.

49

210,

50

0

P e r s s o n , B . , a n d K a r g e r , B.

Chromat.,

, ?I.

Chromat.W.,

Barf,, 451 K * ’ and (1977 Chem.,

23, 473 (1977).

9

9

55. 56

, -J .

Eriksson, B., Andersson, I . , P e r s s o n , B . , A c t a Pharm. Suec. , 1 I t e l l , L . , and Gustafson, A . , Clin.

52

54

505

a n d Edwards, D.

1981).

1 2 , 521 ( 1 9 7 4 ) .

51

53

u-

9

Appel, E . , B a y e r , P . , H a j d u , P., Palm, D., S c h o f e r , J . , a n d U i h l e i n , L’,, Nau-Schm. Arch. Pharmac., 315, 233 (1981). Nimura, N . , K a s a h a r a , Y . , a n d K i n o s h i t a , T . , J . C h r o m a t . , 213, 327 ( 1 9 8 1 ) . Svendsen, H . , and Greibrokk, T . , J . Chromat., 212, 153 (1981). J o h a n s s o n , N . , 2. I&.C h r o m a t . , 4,

(1981)

1435

a n d Young, J . , L i f e S c i . ,

57

Anderson, G . ,

58

Okamoto, K . , I s h i d a , Y . , a n d Asai, K . , J . C h r o m a t . , 167, 205 (1978). Yui, Y . , Kimura, h.. , I t o k a w a , Y. , a n d K a w a i , C . , J . C h r o m a t . , 177, 376 ( 1 9 7 9 ) . Yui,-Y., F u j i t a , T . , Yamamoto, T . , I t o k a w a , Y . , a n d K a w a i , Ch., C l i n . Chem. , 26, 194 (1980). Nimura, N . , I s h i d a , K . , a n d K i n o s h i t a , T . , J . C h r o m a t . , 221, 249 ( 1 9 8 0 ) . Imai, K . , 2. C h r o m a t . , 105, 135 ( 1 9 7 5 ) . Imai, K . , Tsukamoto, N . . , a n d Tamura, Z . , J . S h r o m a t . , 137, 357 (1977) Imai, K . , and Tamura, Z . , Clin. Chem. Acta, 85, 1 ( 1 9 7 8 ) . Yui, Y . , a n d K a w a i , C . , J . C h r o m a t . , 206, 586 (1981) * D a v i s , T . , Gehrke, C . , G e h r k e , C . , J r . , Cunningham, T . , Kuo, K . , G e r h a r d t , K . , J o h n s o n , H . , a n d Willians, C . , C l i n . Chem., 24, 1317 (1978)

59 60. 61. 62. 63 64. 65 * 66.

28, 507 (1981).

I

TERRY D.WILSON

584

67 68. 69 70 71 72 * 73 74

F r o e h l i c h , P . , and Cunningham, T . , Anal. Chim. Acta, 97, 357 (1978). Yui, Y . , Itokawa, Y . , and K a w a i , C . , Anal. Biochem., 108, 11 (1980). Kobayashi, S., S e k i n o , J . , Honda, K . , and Imai, K . , Anal. Biochem,, 112, 99 (1981). K i s s i n g e r , P . , An-em., 49, 447A (1977). K i s s i n g e r , P.! B r u n t l e t t , C . , Davis, G . , Feli c e , J . , Riggin, R . , and Shoup, R . , C l i n . Chem., 23, 1449 (1977). Mefford, I . , Ward, If., F i l e s , L . , T a y l o r , B . , Chesney, b . , Keegan, D . , and Barchas, J . , L i f e S c i . , 28, 477 (1981). K i s s i n g e r , P . , B r u n t l e t t , C . , and Shoup, R., L i f e S c i * I 28, 455 (1981). Refshauge, C., K i s s i n g e r , P . , D r e i l i n g , R . , Blank, L . , Freeman, R . , and Adams, R . , Life 14, 311 (1974). K i s s i n g e r , P . , Kiggin, R . , Alcorn, R . , and Rau, L . , Biochem. Med., 13, 299 (1975). Keller, R X A Y K e f f o r d , I . , and Adams, R e , Life Sci 19, 995 (1976). F e l i c e , L . , F e l i c e , J . , and K i s s i n g e r , P . , J . Neurochem., 31, 1461 (1978). Allenmark, S., and Hedman, L . , J. Liq. Chro-

a.,

75 76

- 0 ,

77

9

78

-* mat

,

2,

277 (1979).

79

S a s a , S . , and Blank, L . , Anal. Chim. Acta,

80.

Asmus, P . , and F r e e d , C . ,

81. 82.

83

134. 29 (1979).

J . Chromat., 169,

303 (1979).

Wagner, J . , Palfreyman, IN,, and Z r a i k a , M . , J . Chromat., 164, 41 (1979). Foyer, T . , and J i a n g , N., J . Chromat., 153,

365 (1978).

9

84. 85. 86. 87 80.

Anderson, G . , B a t t e r , D., w i t z , B . , and Cohen, D . ,

453 (1980).

Kempf, E , , and b a n d e l , P . , 112, 223 (1981).

-

Young, J., ShayChromat., 181,

2.

Anal. Biochem.,

Watson, E . , L i f e -‘I Sci 28, 493 (1981). Allenmark, S,, Hedman, L . , and S o d e r b e r g , A . , Microchem. J 25, 567 (1980). Davis, G., ani’Kissinger, P. , Anal. Chem., (1981). _53, _ _ 156 Shoup, R . , and Kissinger, P . , C l i n . Chem.,

23, 1268 (1977).

LEVARTERENOL BITARTRATE

89. 90.

higgin, R.,

585

and K i s s i n g e r , P . ,

40, 2109 ( 1 9 7 7 ) .

Anal.

Chem.,

91,

Koyer, T., J i a n g , N . , Tyce, G . , and Sheps, S., C l i n . Chem., 2 5 , 256 (1979). Hashimoto, r a n d Paruyama, Y . , J. Chromat.,

92.

Wenk, G . ,

93

L'enner, D . , Brown, I " . , and L h o s t e , F . , J . Chromat., 224, 507 (1981). G o l d s t e i n , D., F e u e r s t e i n , G., I z z o , J . , Kopin, I . , and K r e i s e r , H . , L i f e S c i . , 28,

9

94

152, 387 (1978).

and Greenland, H . ,

183, 261 (1980).

2. Chromat.,

467 (1981).

95

K r s t u l o v i c , A . , Dziedzic, S., Bertani-Dziedzic, L . , and DiHico, D . , 2. Chromat.,

96 97 98. 99

9

100.

101.

217, 523 (1981).

Blank, C., 2. Chromat., 117, 35 (1976). Goto, M., Nakamura, T . , and I s h i i , D . , J . Chromat., 226, 33 (1981). d e s t e r i n k , B . , and Mulder, T . , J. Neurochem ' ' 36, 1449 (1981). O o s t e r h u i s , B . , Brunt, K . , i d e s t e r i n k , B . , and Doornbos, D . , Anal, Chem. , 52, 203

(1980)

Uchikura, K., Horikawa, R . , and Tanimura, T . , J . Chromat., 2 2 3 , 41 (1981). G i t l o w , S., Mendlowitz, M . , B e r t a n i , L . , Wilk, S., and ' d i l k , E . , 2. C l i n , I n v e s t . ,

50, 859 (1971).

102. Maas, J . , and L a n d i s , D., 2. Pharm. Exp. Ther., 177, 600 (1971). b.., Jackman, G . , Bobik, A , , K e l l e h e r , 103. D., J e n n i n g s , G . , Leonard, P . , Skews, H . , and Korner, P . , L i f e -S* )c i 25, 1461 (1979). 104. F i t z G e r a l d , G., Hossmann, V . , Hamilton, C . , Reid, J . , Davies, D . , and D o l l e r y , C . , C l i n . -Pharm. T h e r . , 26, 669 (1979). 105. S i l v e r b e r g , A , , Shah, S . , Haymond, M . , and C r y e r , P . , 2. P h y s i o l . , 234, E252 - (1978). . _ . 106. I\!.aas, J . , Hattox, S , , Greene, N * , and S c i e n c e , 205, 1025 (1979). Landis, D., Black, K O , and 107. Gordon, E., 0-J., Kopin, I . , Biochem. Med ' 11, 32 (1974). 108. S j b q u i s t , €3. Lindstrom,B. , and Anggard, E . , J * Chromat*, 105, 309 (1975).

mer,

&I.

"-0

I.

TERRY D.WILSON

586

109. 110.

111,

Mann, D., L i n c o l n , L . , a n d Yates, P . , L a n c e t , 2 0 , 1366 ( 1 9 8 0 ) . Jackman, G . , C l i n . Chem., 2 6 , 1623 (1980). Anderson, G . , Young, J . , Cohen, D . , Shayw i t z , B., a n d B a t t e r , D . , J . C h r o m a t . , 2 2 2 , 112 (1981).

The p r e s e n t l i t e r a t u r e r e v i e w i n c l u d e s mate r i a l p u b l i s h e d t h r o u g h December, 1981.

MEPROBAMATE Charles M . Shearer

588 588 590 590 590 591

2. Physical Properties 2.7 Crystal Properties 6. Methods of Analysis 6.5 Titrimetric Analysis 6.6 Chromatographic Analysis 7. References

Analytical Profilesof Drug Substances Volume I I

Copyright Q 1982 by The American

587

Phumafeuticd Associalion ISBN 012-260811-9

.

CHARLES M SHEARER

588

The following supplement contains updated information pertaining to the analytical chemistry of meprobamate. A literature survey was conducted and is complete up to January 1981. The numbering system for topics discussed is the same as that in the original profile (Volume 1 , pp. 207-232). 2. Physical Properties

2.2 Nuclear Magnetic Resonance Spectra The carbon-13 NMR spectrum of a Wyeth In-House Reference Standard in deuterated dimethyl sulfoxide as obtained on a Varian FT-80-A spectrometer is presented in Figure 1. The assignment of t e individual signals is given in the following table( 17 . Carbon a b C

d e

f g

Chemical Shift (ppm) 158.39 68.31 37.73 36.84 16.42 15.17 18.99

2.7 Crystal Properties 2.71 Polymorphism Three crystal modifications were investigated by Burger and Schulte (2) and two modifications were investigated by Clements and Popli ( 3 ) . Both references discussed thermal properties, infrared spectra, X-ray diffraction patterns and dissolution studies.

I

I

150

100

Figure 1

wm

J I

50

- 13C NMR Spectrum of Meprobamate

I

0

CHARLES M . SHEARER

590

6. Methods of Analysis 6.5 Titrimetric Meprobamate, upon hydrolysis in acidic solution forms in a stoichiometric amount ammonia, which can be determined, after making solution alkaline, by an ammonia sensing electrode Likewise when meprobamate is hydrolyzed in a basic solution carbon dioxide is formed, which can e determined by a carbon dioxide sensing electrode . 6.64 Column chromatograph Meprobamate has been'determined by HPLC(') using as the eluant varying proportions of chloroform mixed with carbon tetrachloride, hexane o r butyl ether and a column consisting o f small particle fully porous silica packing material or a monomolecular layer of a cyanopropylsilane chemically bonded to a small particle, fully porous silica support. The eluted peaks were monitored using differential refractive index detection.

~$7 .

0)

Meprobamate has also been assayed by HPLC after alkaline hydrolyses, to 2-methyl-2-propyl-l,3propandiol and preparing the benzoyl ester of the di~l(~). An eluant consisting of 60 parts acetonitrile, 30 parts methanol and 30 parts water eluted the benzoyl derivative from a 15 cm x 4.6 mm Ultrasphere RP 18 column in 6 minutes at a flow rate of 2.5 ml/min. This procedure was u s e d for analyses of meprobamate in plasma.

59 1

MEPROBAMATE

7. References 1. Personal communication, B. Hofmann, Wyeth Laboratories, Inc. 2 . A. Burger and K . Schulte, Arch. Pharm. (Weinbaum), 314, 398 (1981). 3. J . A . 3 e m e n t s and S.D. Popli, Can. J . Pharm. Sci., - 8, 88 (1973). 4. Y. Michotte, D.L. Massart and L. Dryon, Pharm. Acta Helv., 52, 152 (1977). 1820 (1979). 5 . S. T z a m i , Chem. Pharm. Bull., 6. I.L. Honigberg, J.T. Stewart and M. Smith, J. Pharm. Sci., 67, 675 (1978). 7 . R.N. G u p E and F. Eng., J. High Res. Chromatogr. and Chromatogr. Comm., 3, 419 ( 1 9 8 0 ) .

I,

TRIAMCINOLONE David H.Sieh

1. Description 2. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.4 Mass Spectra 2.9 SolubilityData 2.10 Crystal Properties 3. Synthesis 5. Drug Metabolism and Pharmacokinetics 5.1 Drug Metabolism 5.2 Pharmacokineticsand Bioavailability 6. Methods of Analysis 6.3 Colorimetric Andysis 6.4 PolarographicAnalysis 6.5 Chromatographic Analysis 6.6 Fluorimetric Analysis 6.7 TitrimetricAnalysis 6.8 DifferentialBorohydride Analysis 6.9 Radioimmunoassay 7. Determinationin Body Fluids and Tissues 8. Acknowledgements 9. References

Analytical PloAlcs of Drup Substances Volume 11

593

594 594 594 594 597 597 598 598 599 599 599 600

600 602 602 609 609 609 609 610 61 1 612

Copfight 0 1981by The A m d w PhUm.ceutial AUodBIion ISBN 0.12.2601)11-9

DAVID H. SIEH

594

The following supplement contains updated information pertaining to the analytical chemistry of triamcinolone. A literature survey was conducted and is complete up to July 1981. The numbering system for topics discussed is the same as that in the original profile (Volume 1, pp. 367-396). 1. Description. Triamcinolone is a glucocorticoid used primarily in the treatment of adrenocortical and rheumatic disorders. 1.13 The CA registry number for triamcinolone is 124-94-7. 2.

Physical Properties 2.1 Infrared Spectra The original profile] contained a comparison of infrared spectral assignments of triamcinolone in mineral oil with that of the compound in the solid state. Bellomonte2 published the infrared spectra (KBr disc) of 13 fluorinated steroids, including triamcinolone and triamcinolone acetonide, and discussed the special influence of the fluorine atom. The major assignments, which are in excellent agreement with Floreyl, are supplemented with the assignments of the C-F str tching frequency; i.e., 1065 (lo) and 979 (2O) cm-f for triamcinolone and 1080 (lo) and 972 (2O) cm-' for triamcinolone acetonide. 2.2

Nuclear Magnetic Resonance The improved 1H-NMR and the 13C-NMR spectra3 of triamcinolone are shown in Figures 1 and 2, respectively (please refer to Figure 4, Section 2.2 of the original profile] for comparison and assignments of the 'H-NMR spectrum). The 13Cspectrum was determined on a JEOL FX60Q spectrometer using a lOmm C/H dual probe. A complete assignment for all the carbon atoms in the 13C-NMR spectrum is listed in Table I. The assignments are based on relative chemical hifts (dimethylsulfoxide-d6 = 39.5 ppm) and l3C-IgF coupling constants and are consistent with literature values for closely related compounds.4 The assignments were simplified by the utilization of polarization transfer to selectively enhance and phase alter individual carbon types.

Figure 1.

'H-Nuclear Magnetic Resonance Spectrum of Triamcinolone ( S Q 9670) in Dimethylsulfoxide-d6. Instrument: Varian XL-100A.

Figure 2.

13C-Nuclear Magnetic Resonance Spectrum of Triamcinolone (SQ 9670) in Dimethylsulfoxide. Instrument: JEOL FX60Q.

597

TRIAMCINOLONE

TABLE I 13C-NMR Spectral Assignmentsa of Triamcinolone Carbon Number 1 2 3

4 5 6 7 8 9 10 11

Chemical Shiftb 152.7 129.1 185.4 124.2 167.0 30.3 27.3 33.3 (19.6) lOl.l(l74.8) 48.0 (22.4) 70.6(37.1)

Carbon Number 12 13 14 15 16 17 18 19 20 21

Chemical Shift 35.9 46.2 43.2 33.6 71.4 87.6 16.7 22.1(5.8) 211.6 66.6

aAll chemical shifts are in ppm from tetramethylsilane (TMS) with internal reference dimethylsulfoxide = 39.5 ppm. bAll spectra were run in DMSO-d6. 13C-19F coupling constants shown in parentheses. 2.4

Mass Spectra The low resolution mass spectra of 28 corticosteroid 21-esters and related compounds of pharmaceutical interest have been determined by Toft and co-workers6 on an AEI MS-12 via direct probe. The mass spectra of triamcinolone and triamcinolone diacetate are in excellent agreement with those published in the original profile. 2.9

Solubility Data 2.91 Solubility Solubilization of 19 steroid hormones, including triamcinolone, triamcinolone acetonide and triamcinolone diacetate by polyoxyethylene lauryl ether was reported by Tomida7 who also concluded that the solubilization of steroids by polyoxyethylene lauryl ether micelles is directly dependent upon their lipophilicity. Triamcinolone was found to have an aqueous solubility of 2.07 x 10-4 M. 2.92

Partition Coefficients The role of crystal structure (as reflected by the melting point and the entropy of fusion)

DAVID H.SIEH

598

and of the activity coefficient (as reflected by the octanol-water partition coefficient) in controlling the aqueous solubility of either liquid or crystalline organic nonelectrolytes, including triamcinolone, triamcinolone acetonide and triamcinolone diacetate was discussed by Yalkowsky and Valvani.lo Structural relationships between a large group of steroids, including triamcinolone and triamcinolone acetonide, and their ether-water partition coefficients were explored by F1ynn.l’ Correlation with biological activity was also discussed. The partition coefficients of triamcinolone in several systems have been reported by Tomida.7 He determined that the aqueous-micellar, octanol-water and ether-water partition coefficients were 96.3, 10.8 and 0.757, respectively. These values have been corroborated by several other investigators.8‘11 2.10

Crystal Properties The mean diamagnetic susceptibility of 20 corticosteroids, including triamcinolone,-was determined by the Faraday method.l2tl3 The orientation of the principal molecular axes with regard to the steroid skeleton was also given. 3.

Synthesis Barton and Heese14 developed and patented a procedure for the chemical conversion of A1-dehydrotriamcinolone to triamcinolone using triphenylmethyllithium in combination with lithium aluminum hydride or LiH2Al(OCH2CH2OCH3)2 at - 7 8 O C. Numerous microbiological dehydrogenations have been published or patented on the conversion of A l dehydrotriamcinolone to triamcino1one.l Maximization of this conversion was reported by Yoshida and co-workers15 using a mixed culture system. Modification and further improvement of the dehydre genation was published16 and patented17 by Ryu and co-workers using a semicontinuous enzymatic process where the concentration of enzyme, active 3-ketosteroid Al-dehydrogenase, is increased in parallel with that of the steroid. Any water-soluble 16,17cycloborate of a steroid of the l6afl7a-dihydroxy3-keto-A4-pregnene series will work in this system.

TRIAMCINOLONE

599

Drug Metabolism and Pharmacokinetics 5.1 Drug Metabolism YamashitaIu studied the metabolism of seven synthetic corticoids, including triamcinolone in rat liver. He found that the metabolism of the corticoids by rat liver slices or homogenates proceeded at rates inversely proportional to the anti-inflammatory potency of the compounds. The major metabolic pathway for triamcinolone was found to be 6B-hydroxylation. In addition, the liver material also metabolized triamcinolone to the 20dihydro and the 11-dihydro derivatives. 5.

Pharmacokinetics and Bioavailability The permeability of the cornea to topically applied drugs, including triamcinolone, has been extensively reviewed by Benson.19 Triamcinolone was usually determined by the blue tetrazolium assay (section 6.31). Local changes in the skin and subcutaneous tissues are known to occur as a result of percutaneous absorption of corticosteroids. This was the subject of an extensive review article by Keipert20 and included discussions of triamcinolone and triamcinolone acetonide. The vasoconstrictor assay was the assay method of choice in these studies. 5.2

The interaction between different corticosteroids, including triamcinolone, and commonly administered antacids and its implication on bioavailability was studied by Naggar, et a1.21 Triamcinolone was found to be quantitatively adsorbed on charcoal and magnesium trisilicate and not adsorbed at all on magnesium or calcium carbonates. The role of bioavailability of steroids on their lipophilic character is of extreme importance. Biagi and co-workers22 showed that the chromatographic Rm value of triamcinolone and several other steroids could be correlated with its lipophilic character. One important conclusion arising from this study is that the dependence of protein binding absorption and biotransformation on lipophilic character might strongly influence the availability of steroids at the site of action. The effects of fourteen steroids, includ-

600

DAVID H . SIEH

ing triamcinolone, on the NMR spectra of liposomes derived from egg yolk phosphatidyl cholines were studied by Ahmad and Mel101-s~~ using continuous wave and Fourier-transform measurements at 60 MHz. The steroids were compared for their ability to broaden the acyl methylene resonances of phosphatidylcholine, when incorporated into liposomes at 25% molar ratio. This study confirmed the importance of the steroid side-chain at C-17 as a requirement for sterol-phospholipid interaction. 6.

Methods of Analysis 6.3 Colorimetric Analvsis .’ 6.31 Occhipinti and c o - ~ o r k e r sreport~~ ed significant improvements in the tetrazolium blue method for the determination of steroids containing an a-ketol side chain. They reported optimal concentration and experimental conditions for rapid quantitative determinations of seventeen representative steroids including triamcinolone and triamcinolone acetonide, occurring in pharmaceutical formulations. Tsuchikura and c o - ~ o r k e r s ~ ~ found that the 1,2-double bond, 16a-hydroxy and 16,17-acetonide groups inhibited the blue tetrazolium reactions of steroids. The stoichiometry and mechanism of the reaction of blue tetrazolium with triamcinolone was studied by Gorag and Horvath.26 On the basis of previous literature data27r28 and experimental results, they concluded that, initially, the side chain was oxidized to the 20-keto-21aldehyde. This oxidation was followed by the basecatalized D-homo rearrangement and subsequent oxidation by blue tetrazolium to form an a-diketone (D ring), which existed in the enolic form. 6.32 Tsuchikura and c o - ~ o r k e r sfound ~~ that the 1,2-double bond, 16a-hydroxy and 16,17acetonide groups inhibited the isonicotinic acid hydrazide reaction of steroids. Yamashita and cow o r k e r ~investigated ~~ the qualitative and quantitative determination of synthetic corticoids and reported the isonicotinic acid hydrazide determination of triamcinolone and triamcinolone acetonide. 6.33 The colorimetric determination of triamcinolone using its reaction with dinitro-

TRIAMCINOLONE

601

phenylhydrazine has been reported.29 6.34 The reaction of a 1 mg/ml aqueous solution of sulfuric acid with triamcinolone produced a yellow color in daylight and no color when viewed with an ultraviolet lamp at 366 nm.30 Triamcinolone did not give a color reaction when treated with vanillin and sulfuric acid.31 6.35 The characterization of glucocorticoids, including triamcinolone, with Dische's reagent (diphenylamine in acetic acid and sulfuric acid) or with a modified Dische's reagent (N-methyldiphenylamine in acetic acid and sulfuric acid) was described by Rioux-Lacoste and Vie1.32 The steroids were dissolved in 2 0 % acetic acid solutions, reacted with the reagent at 85O C. and the absorbance determined spectrophotometrically between 600-650 nm. 6.36 The presence of substituents at C-16 greatly reduces the chromogenicity of 17acorticosteroids in the Porter-Silber test (phenylhydrazine and sulfuric acid).l Although this test has been used for the determination of triamcino1 0 n e 1 2 5 ~ 2it 9 is not recommended because of the = 37 @ 4 2 0 nm) 3 0 low molar absorptivity (Ei:m of the chromogen produced. 6.37 Triamcinolone has been quantitated by its reaction with 2,3,5-triphenyltetrazolium chloride followed by absorbance measurement.33 Triamcinolone was also determined spectrophotometrically with 2,3,5-triphenyltetrazolium chloride by Smoczkiewicz and Jasicziak.34 They found that the absorption of light at 490 nm was much higher €or compounds with neighboring OH groups; i.e., 16a-17u-dio1, than for steroids without an OH group at C-16. A 5 % solution of 2,3,5-triphenyltetrazolium chloride in methanol has been used as a spray reagent for the detection of triamcinolone in thin layer chromatographic systems.34 6.38 A process method has been developed by Ivashkiv3= for rapidly estimating the conversion of 9a-fluoro-16a-hydroxyhydrocortisone to triamcinolone in fermentation broths. The conversion of the steroid was estimated by measuring the absorb-

DAVID H. SIEH

602

ance of the aqueous steroid borate complex at 241.5 and 271 nm and calculating the ratio. 6.4

Polarographic Analysis Differential pulse polarography was used to study the electroanalytical behavior of triamcinolone.37 The half wave potentials vs. the Ag/AgCl electrode of triamcinolone were determined as -1.5OV (C-3 keto reduction) and -1.7OV (C-3 keto reduction) in Britton-Robinson buffer pH 10 (50% v/v in methanol), -1.56V (C-3), -1.75V (C-3) and -1.95 ((2-20 keto reduction) in 0.03M tetramethylammonium hydroxide in methanol and -1.7OV (C-31, -1.98V (C-3) and -2.20V (C-20) in 0.02M tetramethylThis ammonium hydroxide in aqueous DMF (87% v/v). electroanalytical technique was then used to determine triamcinolone in single-component tablets3* and later in multicomponent and complex pharmaceutical preparations.39 6.5

Chromatosraphic Analvsis 6.52 Thi; liyer chroAatographic analysis A compilation of the chromatographic identification and-separation of triamcinolone and other corticosteroids of similar structure is shown in Table 11. This data has been published after 1970. 6.53 Column Chromatography The retention characteristics of 53 steroids on an acetonitrile-diatomaceous earth column are given for elution with n-he tane Triamfollowed by stripping with chloroform. cinolone was recovered nearly quantitatively (98.4%) in the chloroform.

!i3

6.54 High Performance Liquid Chromatography A compiliation of the high performance liquid chromatographic separation systems for triamcinolone published after 1970 is shown in Table 111. ~~

6.55 Gas-Liquid Chromatography Several gas liquid chromatographic systems for the separation and identification of triamcinolone with and without prior derivatization are shown in Table IV. In addition, a gas chromato-

TABLE I1 Thin Layer Chromatographic Systems for the Detection and Determination of Triamcinolone Solvent System Methylene ch1oride:diethyl ether:methanol:water (77: 15 :8 :1 .2)

Adsorbent Kieselgel GF254

Detection UV at 254 nm

Rf 0.15

1,-1-Dichloroethano1:acetone: acetic acid (160:40:10)

0.16

Chloroform Stationary phase-Formamide: acetone (10:90)

0.03

Methylene ch1oride:p-dioxane: water (1 0:5 :5 ) -lower layer

Chloroform saturated with ammonia-methanol (18:l)

Acetone:12N ammonia (99:l)

Silica gel

Kieselgel GF254

Reference 47

Spray with 2,3,5-triphenyl 0.30 -2H-tetrazolium chloride in methanol, heat at 110' C. for 10 minutes. Red-pink color

36

Spray with 5 0 % ethanolic sulfuric acid spray, heat at 120' C. for 5 minutes, UV quench or iodine chamber

48

0.06

0.75

TABLE I1 (continued)

Thin Layer Chromatographic Systems €or the Detection and Determination o f Triamcinolone Solvent System

E

Adsorbent

Detection

R€ -

Ethano1:SN ammonia ( 9 : l )

0.58

Methylene ch1oride:dioxane: water (10 :5 :5) -lower layer

0.40

Ethylene ch1oride:methanol: water (95:5:0.2)

0.05

Methylene ch1oride:dioxane: water (10:5:5)-lower layer

Silica gel 60

UV at 254 nm

Methylene ch1oride:dioxane: water (1 2 :3 :5 ) -lower layer

49

0.07

Butyl acetate or Kieselgel 60 F254 Dich1oromethane:methyl acetate: water (2 :1 :1) -lower layer Stationary phase - Dioxane

Ch1oroform:methanol:glacial acetic acid (90:10:2)

0.22

Reference

Silica gel

Spray with 20% anhydrous Identity zinc chloride in methanol; Test heat at llOo C. for 20 minutes. W at 254 and 366 run

50

UV at 254 nm

51

0.20

TABLE II (continued)

Thin Layer Chromatographic Systems for the Detection and Determination of Triamcinolone Solvent System Chloroform Stationary phase Formamide:acetone (90:10)

Adsorbent Kieselguhr G

Detection Spray with sulfuric acid (1 mg/ml) W at 366 - violet color

Rf

-

0.24

Cyc1ohexane:ethyl acetate: water (25:75:1)

0.15 0.21

Methylene ch1oride:diethylether:water (77:15:8 :1.2)

0.04

Ch1oroform:methanol: acetic acid (9O:lO:Z)

Silica gel

Fluorescence at 2537 Ao

0.20

Reference 30

52

TABLE I11 High Performance Liauid ChromatoeraDhic Seuaration Svstems for Triamcinolone Co1umn

Diol (polar coated silica-5~) 250 x 4 . 5 m ID

Mobile Phase n-hexane:isopropanol (75 : 25)

Flow Rate (ml/min) 1.3

Retention Time Detection (min) or relative (nm) Ref. 20.0 254 54

Bondapak C18 1Ou 30cm x 3.9mm ID

3.3g potassium phosphate dibasic 4.2g potassium phosphate monobasic 2.8L methanol 1.2L water

2.0

1.74

254/280

55

lop Silica gel 25cm x 0.2cm ID

95% ethano1:methylene chloride (5:95)

1.55

4.90

254

56

Spherisorb S10-ODS

methanol :water (56 :44)

1.3 to 1.5

4.5

254

57

Corasil I1 2 ft. x 2.3mm ID

n-hexane:ethyl acetate (3:2)

1.0

9.05

254

58

Bondapak c18/ Corasil 2 ft. x 2.3mm ID

methanol :water (2:3) acetonitri1e:water (1:9) acetonitri1e:water (1 :4)

1.5

0.4 1.0 0.4

TABLE 111 (continued) High Performance Liquid Chromatographic Separation Systems for Triamcinolone Co 1umn

Flow Rate (ml/min)

methanol :water ( 7 0 : 3 0 ) methanol :water (5O:SO) acetonitri1e:water (60:40) acetonitri1e:water (40:60)

1.5

Bondpak C18/ Corasi1 61cm x 2.3mm ID

methanol :water (60:40) methanol :water (40:60) acetonitri1e:water (40:60) acetonitri1e:water (20:SO)

1.5

Bondapak Phenyl/ Corasil 61cm x 2.3mm ID

methano1:water (60:40) methanol :water (40:60) acetonitri1e:water (40:60) acetonitri1e:water (20:80)

1.0

Zorbax SIL 3.8mm ID

cyc1ohexane:methylene ch1oride:ethanol (9:4:1)

Bondapak C18 30cm x 4.0mm ID

p

2

Mobile Phase

Retention Time Detection (min) or relative (nm) Ref. 1.14 1.86

254

59

1.00

50 kg/cm2

1.19 relative to acetone 1.00 1.06 1.76 0.94 1.59 relative to acetone 1.00 0.98 1.56 1.04 1.54 relative to acetone 1.00

28

254

254

60

TABLE IV

Gas Liquid Chromatographic Systems for the Separation and Identification of Triamcinolone

Column

Column T (OC1

Detection Carrier Gas, Flow Rate

Derivatization

Rf I

Ref.

2 . 5 % SE 30 on

250

FID N2, 40 cc/min

None

1% OV-17 on 100/120 silanized Supasorb 9 ft. x 0.25 in.

260

EC N2, 100 cc/min

trimethylsilyl

23.6 m i n .

62

3% OV-17 on Chromosorb WHP 1.8m x 2.0mm

260

F ID He, 60 cc/min

trimethylsilyl

3.53 min

63

80/100 Chromosorb G 2 ft. x 4mm

1.13

Relative to cholesterol 1.00

61

TRIAMCINOLONE

609

graphic analysis of steroids, including triamcinolone, in pharmaceutical products has been reported by Garzo and co-workers.64 6.6

Fluorimetric Analysis Kadinqu has described a fluorimetric determination of several corticosteroids, including triamcinolone and triamcinolone acetonide, containing the A1r4-3-keto group. These A ring dienonecontaining steroids were reacted with zinc dust and 40% sulfuric acid in n-butyl ether. The fluorescence emission and activation maxima were then determined at 390 and 340 nm, respectively. 6.7

Titrimetric Analysis Triamcinolone has been determined by combustion followed by titration of the resulting fluoride ion with thorium nitrate solution and alizarin red S indicator.41 Steroids with a 16a, 17a-diol group can be titrated with lead tetraacetate in acetic acid to a potentiometric endpoint. The micro version of this method using a 0 . 0 0 1 N lead tetraacetate solution enabled triamcinolone contaminant to be determined in its 1 6 , 1 7 - a ~ e t o n i d e , ~ ~ r ~ ~ 6.8

Differential Borohydride Analysis The differential borohydride assay develop ed by Gor6g44 is an excellent method for the determination of A 4 - and A1t4-3-ketosteroids in pharmaceutical preparations. Addition of propylene glycol to the reaction mixture to complex sodium metaborate allowed K i r ~ c h b a u mto ~ ~ improve the reduction of A 4 - and A4r6-3-ketosteroids. However, A1r4-3-ketosteroids, i.e. triamcinolone,were less than 10% reduced with sodium borohydride. By the use of lithium borohydride instead of sodium borohydride, Chafetz and c o - ~ o r k e r swere ~ ~ able to determine triamcinolone. 6.9

Radioimmunoassay A radioimmunoassav .’ for the determination of picogram quantities of triamcinolone in plasma was reported by Loo and Jordan.65 The antibody against triamcinolone was obtained by immunizing rabbits with triamcinolone 21-hemisuccinate coupled to bovine serum albumin. The problem of antiserum cross reactivity (5%)with hydrocortisone was

TABLE V Determination of Triamcinolone in Biological Fluids and Tissues Chromatographic Method

Extraction

GLC with derivatization

Ethyl acetate followed by partitioning between hexane and 70% methanol

GLC with derivatization

Ethyl acetate

Biological System

Internal Standard

Ref. -

Rat muscle

3H-triamcinolone

62

Progesterone

63

Culture media of mouse and human dermal fibroblasts

GLC

Chlor0form

Blood, urine and body tissues

Cholesterol

61

HPLC

Diethylether

Human serum

Prednisone

54

Dichloromethane

Plasma and urine

TLC with fluorescence

5

H-triamcinolone 51,52

TRIAMCINOLONE

611

circumvented by the transformation of the steroids to their Girard T hydrazones in ethanol denatured plasma. Triamcinolone reacted very slowly with Girard T. Determination in Body Fluids and Tissues Triamcinolone has been determined in bioloqical fluids and tissues by several different methods of analysis (see Section 6 . 9 ) in combination with various extraction techniques. Table V summarizes the results that have been published since 1 9 7 0 . In addition, Saito and co-workers60 have described a high pressure liquid chromatographic method for the separation and quantitation of 12 synthetic corticosteroids in blood and urine. Triamcinolone administration produced serum peaks at 2-5 hours with t+ = 4 hours. 7.

Acknowledgements The author would like to express his appreciation to Mr. Steve Highcock for performing the literature search, to Dr. Michael Porubcan for determining and analyzing the nuclear magnetic resonance spectra, to Dr. John Dunham for his critical review of the manuscript, and to Marie Bruno for typing the manuscript. 8.

DAVID H.SIEH

612

9.

References

1. Florey, K., Analytical Profiles of Drug Substances, Vol. 1, Academic Press, New York, N.Y., pp. 367-396 (1972). 2. Bellomonte, G., Ann. 1st. Super. Sanita, g(2-31, 121-128 (1976). 3. Porubcan, M., Squibb Institute for Medical Research, communication. . personal 4. Blunt, J.W.; Stothers, J.B.,Org. Magn. Res., 9 (8), 439-444 (1977) 5. Doddrell, D.M.; Pegg, D.T.,J. Amer. Chem. SOC., 102, 6388-6391 (1980). 6. Toft, P.; Lodge, B.A.; Simard, M.B., Can.J. Pharm. Sci. , 7(2) , 53-61 (1972) 7. Tomida, H.; Yotsuyanagi, T.; Ikeda, K., Chem. Pharm. Bull. , 26(9) , 2832-2837 (1978). 8. Grady, L.T.; Hayes, S.E.; King, R.H., Klein, H.R.; Mader, W.J.; Wyatt, D.K.; Zimmerman, R.O., Jr., J. Pharm. Sci., 62(3) , 456-464 (1973). 9. Verma, S.C.; Runikis, J.O.; Stewart, W.D., Indian J. Hosp. Pharm. , lO(5) , 167-173 (1973). 10. Yalkowsky, S.H.; Valvani, S.C.; J. Pharm. Sci., 69 (8), 912-922 (1980). 11. Flynn, G.L. , ibid. , 60(3), 345-353 (1971). 12. Van den Bossche, G.; Sobry, R., Acta Crystallo., Section A. , A31(3) , 318-322 (1975). 13. Van den Bossche, G., _Zl Kristallogr. Kristallgeom., Kristallphys., Kristalchem., 136(5-6) , 402-410 (1972); C.A. 78:148114. 14. Barton, D.H.R.; Heese, R.H., Ger. Offen. 2329729 (1974); C.A. 80:108758. 15. Yoshida, T.; Sueki, M.; Taguchi, H.; Kulprecha, S.; Nilubol, N.; Eur. J. Appl. Microbiol. Biotechnol., 11, 81-88 (1981). 16. Ryu, D.Y.; Lee, B.K.; Thoma, R.W.; Humphrey, A.E., Chem. Eng. Progr., Symp. Ser., 67(108) , 80-84 71971) 17. Ryu, D.Y.; Lee, B.K.; Thoma, R.W., U.S. Patent 3585110 (1971). 18. Yamashita, O., Nippon Naibumpi Gakkai Zasshi, 47(12) , 1033-1045 (1972); C.A. 77:83767. 19. Benson, H., Arch.Ophthalmol., 91, 313-327 (1974). 20. Keipert, J.A., Med. J. Aust., 1(19), 1021-1025 (1971). 21. Naggar, V.F.; Gouda, M.W.; Khalil, S.A., Pharmazie, 32(2), 778-781 (1977).

.

613

TRIAMCINOLONE

22. Biagi, G.L.; Barbaro, A.M.; Gandolfi, 0.; Guerra, M.C.; Cantelli-Forti, G., J. Med. Chem., 18(9) , 837-838 (1975). 23. Ahmad, P.; Mellors, A., J. Membr. Biol., 41(3), 235-247 (1978). 24. Occhipinti, E.; Rigamonti, G.; Calati, M., Farmaco, Ed.Prat., 29(11) , 611-620 (1974). 25. Tsuchikura, H.; Shimano, H.; Uete, T., Horumon To Rinsho, 18 (6), 501-504 (1970); C.A. 73:66805. 26. Gzrog, S.; Horvath, P., Analyst, 103(1225), 346-353 (1978). 27. Graham, R.E.; Biehl, E.R.; Kenner, C - T - ; Luttrell, G.H. ; Middleton, D.L. ; J. Pharm. Sci., 64, 226-230 (1975). 28. Graham, R.E.; Biehl, E.R.; Kenner, C.T., -ibid., 65, 1048-1051 (1976). 29. Yamashita, 0.;Mineo, Y.; Nakagawa, T.; Matsuoka, K.; Nakamura, M.; Mishina, Y.; Marumoto, S.; Okada, K., Horumon To Rinsho, 19 (7), 531-535 (1971): C.A. 77:16241. 30. Vanderhaeghe, H.; Hoebus, J.; J. Pharm. Belg., 31(1) , 25-37 (1976). 31. Hiai, S.; Oura, H.; Nakajima, T.; Planta Med., 29 (2), 116-122 (1976) 32. Rioux-Lacoste, C.; Viel, C., Ann. Pharm. Fr., 33 (3-4), 163-170 (1975). 33. Heintz, B.; Kalusa, R., Dtsch. Apoth.-Ztg., 118 (27), 1000-1006 (1978). 34. Smoczkiewiczowa, A. ; Jasiczak, J. , Chem.Anal., 16 (5), 1091-1099 (1971). 35. Ivashkiv, E., Biotechnol. Bioeng., 13(4) , 561-567 (1971). 36. Byrne, J.A.; Brown, J.K.; Chaubal, M.G.; Malone, M.H., J. Chromatogr., 137 (2), 489-492 (1977). 37. DeBoer, H.S., Den Hartigh, J.; Ploegmakers, H.H.J.L., Van Oort, W.J., Anal.Chim.Acta., 102, 141-155 (1978). 38. DeBoer, H.S.; Lansaat, P.H.; Kooistra, K.R., Van Oort, W.J., ibid., 111(1) , 275-279 (1979). 39. DeBoer, H.S.; Lansaat, P.H., Van Oort, W.J., ibid., 116 (11, 69-76 (1980). 40. Kadin, H., Microchem. J., 20(2), 236-241 (1975). 41. Kofman, M.D.; Arzamastsev, A.P., Farmasiya, 21(3) I 25-27 (1972). 42. Czizier, E.; Garb'g, S.; Tzen, T., Microchim. Acta, 966 (1970).

.

DAVID H.SIEH

614

43. 44. 45. 46. 47.

Gorijg, S., "The Analysis of Steroids," CRC Crit.Rev.Anal.Chem. , 9 ( 4 ) , 3 3 3 - 3 8 3 ( 1 9 8 0 ) . Ggrbg, S., J. Pharm. Sci., 5 7 , 1 1 3 7 - 1 1 4 0 ( 1 9 6 8 ) . Chafetz, L.; Tsilifonis, D.C.; Riedl, J.M., ibid., 61, 1 4 8 - 1 5 0 ( 1 9 7 2 ) . Kirschbaum, J. , ibid, 6 7 ( 2 ) , 2 7 5 - 2 7 6 ( 1 9 7 8 ) . Cavina, G.; Chemello, N.; Loberto, D.; Rocchi, I.; Romaniello, E.; Schweiger, L.; Zanni, G., Ann. 1st. Super. Sanita, 9 ( 4 ) , 261-309

(1973).

166(1)

299-304

48.

Bailey, K.; By, A.W.; Lodge, B.A., J.Chromatog.,

49.

Lanouette, M.; Lodge, B.A., ibid., 1 2 9 ,

I

(1978).

475-477 ( 1 9 7 6 ) . 5 0 . Martin, J.L.; Duncombe, R.E.; Shaw, W.H.C., Analyst, 100 ( 1 1 8 9 ) , 2 4 3 - 2 4 8 ( 1 9 7 5 ) . 51. Kusama, M.; Sakauchi, N.; Kumaoka, S., Metab. , Clin. Exp. , 2 0 ( 6 ) , 5 9 0 - 5 9 6 ( 1 9 7 1 ) 52. Kusama, M.; Sakauchi. N.: Kumaoka. S . . Nitmon Naiburnpi Gakkai Zasshi, 4 6 ( 6 ) , 6 5 4 - 6 5 8 (1'970). 53. Graham, R.E.; Kenner, C.T., J. Pharm. Sci., 6 2 (11), 1 8 4 5 - 1 8 4 9 ( 1 9 7 3 ) . 54. Schoeneshoefer, M.; Skobolo, R.; Dulce, H.J., J. Chromatogr. , 2 2 2 ( 3 ) , 4 7 8 - 4 8 1 ( 1 9 8 1 ) . 55. Baker, J.K.; Fifer, E.K., J. Pharm. Sci., 6 9 ( 5 ) , 590-592 ( 1 9 8 0 ) . 56. Gaetani, E.; Laureri, C.F., Farmaco, Ed.Prat., 2 9 ( 2 ) I 110-118 ( 1 9 7 4 ) . 5 7 . Gordon, G.; Wood, P.R., Analyst, 1 0 1 ( 1 2 0 8 ) , 876-882 ( 1 9 7 6 ) . 58. Hara, S.; Hayashi, S., J.Chromatogr., 1 4 2 , 689-703 ( 1 9 7 7 ) . 59. Tymes, N.W., J.Chromatogr. Sci., 1 5 ( 5 ) , 151-155 (1977). 60. Saito, Z . ; Amatsu, E.; Ono, T.; H i h u m i , S.;

.

Mimou, T.; Hashiba, T.; Sakato, S . ; Miyamot0,M.i Takeda, R., Nippon Naibumpi Gakkai Zasshi, 55(10)

,

1296-1306

(1979).

Finkle, B.S.; Cherry, E.J.; Taylor, D.M., J.Chromatogr.Sci. , 9 ( 7 ) , 3 9 3 - 4 1 9 ( 1 9 7 1 ) . 6 2 . Simpson, P.M., J.Chromatogr., - 7 7 ( 1 ) , 161-174

61.

(1973). 63. Au, D.S.-L.; Runikis, J.O.; Abbott, F . S . ; Burton, R.W.; J.Pharm.Sci. , 7 0 ( 8 ) , 9 1 7 - 9 2 3 ( 1 9 8 1 ) . 64. Garzo, G.; Blazso,M.; G6rijg, S., Proc.Conf.App1. Phys.Chem. ,2nd. , 1, 3 0 1 - 3 0 6 ( 1 9 7 1 ) i CA 7 6 : 9 0 0 8 8 . 6 5 . Loo, J.C.K.; Jordan, N., Res.Commun.Chem.Patho1. Pharmacol. , 2 3 ( 3 ) , 4 9 3 - 5 0 4 ( 1 9 7 9 ) .

TRIAMCINOLONE ACETONIDE David H. Sieh

1. Description 2. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.4 Mass Spectra 2.9 Solubility Data 2.10 Crystal Properties 3. Synthesis 5. Drug Metabolism and Pharmacokinetics 5.1 Drug Metabolism 5.2 Pharmacokinetics and Bioavailability 6. Methods of Analysis 6.2 Direct Ultraviolet Analysis 6.3 Colorimetric Analysis 6.4 Polarographic Analysis 6.5 Chromatographic Analysis 6.6 Fluorimetric Analysis 6.7 Titrimetric Analysis 6.8 Differential Borohydride Analysis 6.9 Radioimmunoassay 7. Determination in Body Fluids and Tissues 8. Acknowledgements 9. References

615

616 616 616 616 619 620 62I 622 623 623 624 627 627 627 629 629 641 641 641 642 642

644 644

Copyright 0 1982 by The American Pharmaceutiul Amcialion ISBN 012-260811-9

DAVID H . SIEH

616

The following supplement contains updated information pertaining to the analytical chemistry of triamcinolone acetonide. A literature survey was conducted and is complete up to July 1981. The numbering system for topics discussed is the same as that in the original profile (Volume 1, pp. 397-422).

1.

Description

Triamcinolone acetonide is a glucocorticoid used mainly in the treatment of adrenocortical and rheumatic disorders. 1.13 The C A Registry Number for triamcinolone acetonide is 76-25-5. 2.

Physical Properties 2.1

Infrared Spectra

The original prof ilel contains a comparison of infrared spectral assignments of triamcinolone acetonide in mineral oil with t3at of the compound in the solid state. Bellomonte has published the infrared spectra (potassium bromide disc) of 13 fluorinated steroids, including triamcinolone and triamcinolone acetonide, and discussed the special influence of the fluorine atom. The major assignTents, which are in excellent agreement with Florey , are supplemented with the assignments of the C-F streFching frequency i.e., 1080 (1') and 972 (2') cm for triamcinolone acetonide. Triamcinolone acetonide has been identified by a combinaf ion of optical rotation and infrared spectroscopy

.

2.2

Nuclear Magnetic Resonance Spectra

The improved 'H-NMR and the I3C-NMR spectra4 of triamcinolone acetonide are shown in Figures 1 and 2, respectively (refer $0 Figure 2, Section 2.2 of the originallprofile for comparison and signments of the H-NMR spectrum). The "C-spectrum was determined on a JEOL FX60Q spectrometer using a 10 mm C/H dual probe. A compi5te assignment for all the carbon atoms in the C-NMR spectrum is listed in Table I. The assignments are based on relative chemical shifts

a,

a

-d

a,

d 0 c,

u a

k

a,

Figure 2.

13C-Nuclear magnetic resonance spectrum of triamcinolone acetonide (SQ 9727) in dimethylsulfoxide-d6. (Instrument: JEOL FX60Q).

619

TRIAMCINOLONE ACETONIDE

(dimethylsulfoxide - d6 = 39.5 PPM) and 13C-”F coupling constants and are consistent with 5 literature values for closely related compounds The assignments were simplified by the utilization of polarization transfer to selectively6enhance and phase alter individual carbon types

.

.

Table I. Carbon Number

I3C-NMR Spectral Assignmentsa of Triamcinolone Acetonide Chemigal Shift

Carbon Number

Chemical Shift

13 1 44.7 152.4 14 2 42.8 129.0 15 185.1 3 33.0 124.2 16 4 80.8 5 166.5 17 97.2 18 6 30.1 16.3 19 7 22.7 (5.8) 27.5 20 32.5 (16.6) 8 209.7 100.9 (175.8) 9 65.9 21 d 10 47.3 (22.4) 110.4 -C-e 26.3 11 70.4 (36.1) CH,J 12 36.6 25.3 a All chemical shifts are in PPM from tetramethylsilane with intepal reference dimethylsulfoxided = 39.5 gPy3 19All spectra were determined in DhSO-d g- F coupling constants shown in parentkeses. Methylene carbon of the acetonide. Methyl groups of the acetonide.

.

2.4

Mass Spectra

The low resolution electron impact mass spectra of several fluorinated corticosteroidal 1,4-giene-3-ones have been determined by Lodge and The mass spectral fragmentation pattern Toft found for triamcinolofe acetonide is in excellent agreement with Florey In addition, the mass spectra of triamcinolone acetonide 21-t-butylacetate was determined for comparison purposes. The gas chromatographic-mass spectral characteristics of silanized triamcinolone

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620

DAVID H. SIEH

.

acetonide were determined by Painter8 Under the reaction conditions employed (see section 6.55 for reaction conditions and gas chromatographic details), triamcinolone acetonide was initially silanized at the C-21 position (m/z 506) and much slower at the C-11 position (m/z 5 7 8 ) . An intense fragment at m/z 4 4 7 [ l o s s of COCH OSi(CH ) 3 from the disilanized material (m/z 578f helped 2onfirm the structure. 2.9

Solubility Data 2.91 Solubility

Solubilization of 19 steroid hormones, including triamcinolone, triamcinolone acetonide and triamcinolone diacetate, by polyoxyethylene lauryl ether was reported by Tomida who also concluded that this solubilization is directly dependent upon the lipophilicity of the steroids. Triamcinolone acetonide yas found tg have a solubil$.by of 4 . 9 5 x 10- M in water , 20 mg/g in acetone , f1012 mg/ml in water and 1.44 mg/ml in 4 0 % ethanol Dissolution rates of triamcinolone acetonide in aqueous media werf2determined at 28OC via a rotating-disc method Triamcinolone acetonide solubility in distilled water ranged from 21 mcg/ml at 28OC to 33.6 mcg/ml at 5OOC. The dissolutio con tant at maximum agitation was -9 rat? 4 . 5 1 x 10 hr- cm-9 Solubilities and dissolution rates were markedly lower in potassium chloride solution. The blue tetrazolium assay (section 6.31) was used to determine the triamcinolpge acetplqide. When the same laboratory used C-triamcinolone acetonide to determine solubilities, lower values were obtained. Triamcinolone acetonide solubility in distilled water ranged from 1 7 . 5 mcg/ml at 28OC to 26.5 mcg/ml at 5OOC.

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.

2.92 Partition Coefficients

The partition coefficients of triamcinolone acetonhde in 3 systems have been determined by Tomida He determined that the aqueous-micellar, octanol-water and ether-water partition coefficients were 569, 2051 and 14.6, respectively. The values of the octanol-water and

.

'TRIAMCINOLONEACETONIDE

62 1

ether-water partition coefficients are in excellent agrffyf@flyith those published by other investigators The role of crystal structure (as reflected by the melting point and the entropy of fusion) and of the activity coefficient (as -reflected by the octanol-water partition coefficient) in controlling the aqueous solubility of either liquid or crystalline non-electrolytes, including triamcinolone, triamcinolone acetonide and triamcinolone diacetaff, was discussed by Yalkowsky and Vaiyani Fairbrother found that a partition system between acetonitrile and n-hexane gave quantitative separation of triamcinolone acetonide, found in the acetonitrile phase, from petrolatum. Structural relationships between a large group of steroids, including triamcinolone acetonide, and their ether-water paftition coefficients were explored by Flynn Correlation with biological activity was also discussed. The correlation of partitioning and percutaneous absorption of topically applied triamcin93one acetonide has been studied by Lien Partition coefficients of and Tong triamcinolone acetonide on f6diatomaceous earth column have been determined (see section 6.56).

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.

.

2.10 Crystal Properties

Powder X-ray diffraction d a t a '' indicate that triamcinolone acetonide may exist in two polymorphic forms. For example, polymorphic form 1 exhibited a characteristic peak at 6.1 A', often split with a smaller peak at 5.95 A'. Polymorphic form 2, on the other hand, showed a large 6.3 A' peak but none at 5 . 9 5 or 6.1 A'. Several other differences in the diffractograms were also noted. Infrared spectroscopy and differential scanning colorimetry were unable to differentiate between the two polymorphs. Several parameters of grain size distribution and particle nature of triamcinolone acetonide crystal suspensions were determined by polarization, electrof8transmission and electron scanning microscopies

.

622

DAVID H. SIEH

The sizes of drug particles in eye ointment suspensions, including triamcinolone acetonide preparationggwere measured microscopically by List and Groenig using a membrane filtering technique. Murata and co-workers2' have reported the observation of crystal forms of triamcinolone acetonide, triamcinolone diacetate and other glucocorticoids in sterile aqueous suspension by electron microscopy. 3.

Synthesis

Acetylation of 9a-fluoroprednisolone-21acetate followed by deacetylation of the triacetate using potassium acetate in dimethylformamide at 11O-12O0C for 4 hours under a nitrogen atmosphere gave 9a-fluoro-pregna-1,4,16triene-11~,21-diol-3,2O-dione diacetate. The resulting diacetate was oxidized with aqueous potassium permanganate in acetone-formic acid for 3 seconds in a flow system at -5OC to give 1511-hydroxyprednisolone in 90% yield. Ketalization with acetone followed by treatment with sodium hydroxide in methanol at O°C for 3 hours in 91:itrogen atmosphere gave triamcinolone acetonide Toth and A o - ~ o r k e r spatented ~~ a process where triamcinolone acetonide 21-nitrate was treated with hydrogen fluoride in chloroform f o r 3 hours at O°C to give triamcinolone acetonide in an 83% yield.24 Reyba has patented a general procedure for the synthesis of acetal fluoro derivatives of steroids. For example, 5 grams of triamcinolone in 8 ml of acetone was treated with 2 ml of perchloric acid and stored at 15-17OC with agitation for 2 hours to give 3.31 grams of triamcinolone acetonide. Castelli and A ~ c h e r ideveloped ~~ and patented a one-step transformation of 16a,l7a-dihydroxy9,11-epoxy-3-oxo-pregn-1,4-diene to triamcinolone acetonide. In a typical synthesis, one equivalent of acetone was added to a 50% hydrogen fluoride solution cooled to -3OOC followed by the addition of one equivalent of the epoxide. Microbial transformation of 16a,l7a-dihydroxy-3-oxo-pregn-4-ene-16,17-acetonide to

TRIAMCINOLONE ACETONIDE

623

triamcinolone acetonide was accomplished by incubatign with Arthrobacter simplex for 12 hours at 28OC A procedure for the synthesis of 16,17dihyroxy steroid acetal and ketal derivatives, including triamcinolone acetonide and2friamcinolone diacetate, has been patented

.

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5.

Drug Metabolism and Pharmacokinetics 5.1

Drug Metabolism

Since 68-hydroxylation was found to be the major metabolic pathway for triamcinolone in vitro and in vivo, this pathway was suggested for the metabolisy80f triamcinolone acetonide. Kupfer and Partridge found that the post mitochondria1 supernate of rat liver homogenate, supplemented with NADPH, hydroxylates triamcinolone acetonide with 68-hydroxytriamcinolone acetonide being the major metabolite. Inducers of the hepatic microsomal monooxygenase system, phenobarbital and l-benzyl-2-thio-5,6-dihydrouracil, enhanced the 6B-hydroxylatiyj. Yamashita found that the metabolism of triamcinolone acetonide by rat liver slices or homogenates proceeded at rates inversely proportional to the antiinflammatory potency of the compound. The liver material metabolized triamcinolone acetonide to 68-hydroxytriamcinolone acetonide. Since esterification of steroid alcohols is known to have a marked influence on their fgsorption and excretion, the metabolism of C-triamcinolone acetonide 21-phosphate in dogs, monkeys angorats was studied by Kriaelani and They found that the C-labelled co-workers steroid was completely hydrolyzed to triamcinolone acetonide following intramuscular or intravenous injection. The radioactivity was eliminated rapidly (t = 1 to 2 hours) from plasma and tissues an8 the major route of excretion was via the bile. 68-Hydroxytriamcinolone acetonide was found to be the major metabolite in urine of all three species. A l s o , metabolites present in the urine as glucuronides and sulfates accounted for approximately 10-15% of the radioaSiivity. Recently, Gorqgn and Morrison studied the metabolic fate of C-triamcinolone acetonide in

.

DAVID H.SIEH

624

rabbits, dogs, monkeys and rats. In the dog, rat and monkey the major excretory route was the feces irrespective of the mode of administration. In the rabbit the excreted radioactivity was equally distributed between urine and feces. The metabolites were isolated by preparative TLC, located by autoradiography and eluted and analyzed by MS, IR, UV and NMR. The major metabolites of triamcinolone acetonide were identified as the C-21 carboxylic acids of triamcinolone acetonide and of 6B-hydroxytriamcinolone acetonide and the previously identified 6B-hydroxytriamcinolone acetonide. In addition, MS and W data indicated the presence of 9a-fluoro-11B,16a,l7-trihydroxy-3,20dioxo-1,4,6-pregnatrien-21-0ic acid cyclic 16,17acetal. They were unable to find conjugation as a major mechanism of excre3i:gj Zimmerman and Bowen studied the teratogenic effects of triamcinolone acetonide on mice by monitoring the incidence of cleft palate. Since practically all of the radioactivity found in the embryos and placentas was unmetabolized drug, it was concluded that the triamcinolone acetonide and not its metabolites was the teratogenic agent. 5.2

Pharmacokinetics and Bioavailability

Since the desired site of activity for topical corticosteroids is the dermis or the epidermis, percutaneous absorption of these materials has been extensively studied. Local changes in the skin and subcutaneous tissue caused by percutaneous absorption was the Qybject of an extensive review article by Keipert and included discussions of triamcinolone and triamcinolone acetonide. Kukita and c o - ~ o r k e r sreported ~~ that triamcinolone acetonide was absorbed percutaneously mainly by3the transfollicular route. Hartmann and Ude studied the effect of triamcinolone acetonide treatment with occlusive foil by monitoring the plasma cortisol, urinary cortisol and urinary triamcinolone acetonide through quantitative thin layer chromatography. Plasma cortisol and urSqary cortisol values were also used by Rasmussen to study the percutaneous absorption of triamcinolone acetonide in a 0.1%

625

TRIAMCINOLONE ACETONIDE

ointment. Serum cortisol values were measured by a radioallergosorbent test. Bioavailability and activity of triamcinolone acetonide using a novel drug delivery system, the aeresol qyAck-break foam, was studied by Woodford In another study, Woodford and and B35ry Barry compared the bioavailability and activity of 0.1% triamcinolone acetonide and 0.1% amcinonide preparations to a 0.025% fluocinoline acetonide gel using a xasoconstrictor assay. Vericat and co-workers evaluated the bioavailability of triamcinolone acetonide following topical application as a suspension in oleoaqueous cream, suspension in fat excipient or polyalcoholic solution by a vasoconstrictor assay. Greatest bioavailability was observed with the alcoholic solutions of the compound. A method using the vasoconstrictive properties of corticoids as indicated by thermal conductivity to measure the amount and speed of these compounds, including triamcinolone acetonide, penetrating the skin aftfly topical application was described by Elimination biokinetics of 0.01, 0.10 Tronnier and 1.0% triamcinolone acetonide creams using a vasoconstgjctor assay has been reported by Barry and Brace Quantitative determination of percutaneous 3bsorption of radiolabeled drugs, including H-triamcinolone acetonide, was described by Schaeffet3agq co-workers in a series of articles Using autoradiographic techniques, a rapid penetration into the living layers of the skin was observed. However, total excretion in the urine took more than 72 hours after removal of the excess of substance from the skin. Autoradiographic techniques were also used to guantitate the percutaneous absorption o i 9 - 5 1 H-triamcintg:gG acetonide in human skin I and in huma32;5jn from lanolin animal skin Penetration of alcohol-ethyl cellulose film triamcinolone acetonide through epidermal membranes was ggeatly enhanced by the addition of salicylic acid The influence of keratolytics and moisturizers on the bioavailability of triamcinolone acetonide using the blanching effect as the parameter of5getection was examined by Gloor and Lindemann

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.

DAVID H. SIEH

626

Verma and co-workers'' compared the potency and effectiveness of fluorinated steroids like triamcinolone acetonide and nonfluorinated steroids such as 16a-hydroxyprednisolone acetonide by studying solubility, partition coefficients, vasoconstrictor ability and penetration across human epidermis. They concluded that the 9a-fluoro group has no effect on the potency of triamcinolone acetonide. The correlation of physicochemical properties i.e., lipophilicity as measured by partitioning in suitable solvent systems, molar refraction, Taft's polar substituent constant ( a o ) , molecular weight and water solubility, and percutaneous absorption of topically applied drugs, including triamcino39ne acetonide, has been studi9j by Lien and Tong Biagi and co-workers showed that the chromatographic R value of triamcinolone acetonide could bg correlated with its lipophilic character. Therefore, the dependence of protein binding absorption and biotransformation on lipophilic character strongly influences the availability of steroids at&he sight of action. Hackney and co-workers demonstrated that mouse fibroblasts growing in vitro contain a binding component for triamcinolone acetonide which is apparently distributed intracellularly, largely as a cytoplasmic soluble macromolecule. Furthermore, the structure-activity relationships of steroids active in growth inhibition were found to be exacjtly paralleled by their ability to displace H-triamcinolone acetonide from this binding component. A new, selective drug delivery system, which uses liposomes as drug carriers, for the topical route of administratiog-,was recentljy described by Using C-triamcinolone Mezei and Gulasekharam acetonide, they found that liposomal encapsulation increased the concentration of the drug at the site where its activity is desired i.e., epidermis and dermis. Because of the selectivity of the liposomal dosage form, it could provide increased efficacy and decreased toxicity of topically, applied drugs.

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627

TRIAMCINOLONE ACETONIDE

6.

Methods of Analysis 6.2

Direct Ultraviolet Analysis

In the ultraviolet spectrum of a mixture of triamcinolone acetonide and chlorhexidine, the overlapping absorption maxima were approximated by the use of Gauss-Lorenz function paraEtters for calculations on a programmed computer Polysorbate 80 and methyl- and propyl-p-hydroxybenzoates were found to interfere in the determination of corticosteroids such as triamcinolone acetonide in dermatological preparatigfs by the ultraviolet spectrophotometric method

.

.

6.3

Colorimetric Analysis

6.31 Ochipinti and co-workers62 reported significant improvements in the tetrazolium blue method for the determination of steroids containing an a-ketol side chain. They reported optimal concentration and experimental conditions for rapid quantitative determinations of 17 representative steroids, including triamcinolone and triamcinolone acetonide, occuring in pharmaceutical fggmulations. Tsuchikura and co-workers found that the 1,2-double bond, the 16a-hydroxy and the 16,17-acetonide groups inhibited the blue tetrazolium reaction. Notable interference was shown by polyethylene glycols, propylene glycol and lanolin in the determination of corticosteroids such as triamcinolone acetonide in dermatological 61 preparations by the blue tetrazolium method Sorbitol and squalene caused only slight interference. A reaction rate method using a modified blue tetrazolium reaction for the determination68f triamcinolone acetonide has been described This method depends on the mixing of a 5% tetramethylammonium hydroxide in absolute ethanol solution with a blue tetrazolium solution of triamcinolone acetonide by an automatic stoppedflow system and monitoring the absorbance at 525 nm during selected measurement time.

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.

6.32 Tsuchikura and c o - ~ o r k e r sfound ~~ that the 1,2-double bond, the 16a-hydroxy and the

DAVID H . SIEH

628

16,17-acetonide groups inhibited the isonicotinic acid hydrazide6seaction of steroids. Yamashita and co-workers investigated the quantitative and qualitative determination of synthetic corticoids and reported the isonicotinic acid hydrazide determination of triamcinolone and triamcinolone acetonide. Substances including carbonyl, reducing,acidic and basic compounds interfered with the determination of triamcinolone acetonide in ointment prepggations by the isonicotinic acid Girard’s fractionation method hydrazide method was used to remove the interfering substances prior to measurement.

.

6.33 The reaction of a 1 mg/ml aqueous solution of sulfuric acid with triamcinolone acetonide produced a faintly yellow color in daylight and no color when@ewed with an ultraviolet lamp at 366 nm

.

6.34 Although the Porter-Silber test (phenylhydrazine and sulfuric acid) has been used for the dtfermination of triamcinolone acetonide , it is not6yecommended because of the low molar absorptivity of the chromogen produced. 6.35 The reaction of triamcinolone acetonide with 2,3,5-triphenyltetrazolium chloride instead of tetrazolium blue followed by absorbance measurement according to the European Pharmacopeia was used to quantitate the $&eroid in pharmaceutical preparations

.

6.36 A new method based on the oxidation of the C-17 side chain with cupric acetate, condensation of the resulting 20-keto-21-aldehyde with 4,5-dimethyl-o-phenylenediamine and spectrophotometric measurement of the quinoxaline derivative gbtained has been described by Szepesi and Gdrdg for the determination of 21-hydroxycorticosteroids, including triamcinolone acetonide, in pharmaceutical preparations. This procedure was used for the analysis of bulk material (purification on a cation exchange column was required after the oxidation step) or ointment (no purification required). Chafetz and Tsilif~nis’~ also used a cupric acetate oxidation step to determine triamcinolone

629

TRIAMCINOLONE ACETONIDE

acetonide. The oxidation step was followed by chromogen formation with aqueous acidic phenylhydrazine and absorption measurement at 370 nm. This procedure is claiE5d to be twice as sensitive as STfpesi and Gdrdgs Bundgaard quantitatively determined triamcinolone acetonide by cupric acetate oxidation followed by condensation with 3-methyl-benzothiazol-2-one hydrazone in alkaline solution to form the highly absorbing azine (Xmax = 400 nm). This method was used to determine triamcinolone acetonide in the presence of its 21-acetate ester.

.

6.4

Polarographic Analysis

Differential pulse polarography was used to study the elecf5oanalytical behavior of triamcinolone acetonide The half-wave potentials vs Ag/AgCl electrode of triamcinolone acetonide were determined as -1.46 V (C-3 keto reduction) and -1.70 V (C-3 keto) in Britton-Robinson buffer pH 10 ( 5 0 % v/v in methanol), -1.60 V ((2-3 keto), -1.79 V (C-3 keto) and -2.03 V (C-3 keto) in 0.03 M tetramethylammonium hydroxide in methanol and -1.71 V (C-3 keto) , -1.98 V (C-3 keto) and -2.15 V (C-20 keto) in 0.02 M tetramethylammonium hydroxide in 87% aqueous dimethylformamide. This electroanalytical technique was then used to determine triamcinolone acetonide in multicomponent and complex pharmaceutical preparations73

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.

6.5

Chromatographic Analysis 6.52 Thin Layer Chromatographic Analysis

A compilation of the thin layer chromatographic properties and identification of triamcinolone acetonide published after 1970 is shown in Table 11. IdentificatifB30f triamcinolone acetonide in a 0.025% ointment was accomplished by thin layer chromatography on silica gel GF plates using a developing solvent of methanol: chloroform: benzene (20:100:40) and a 0.2%7Q1ue tetrazolium in methanol spray reagent. Massa identified triamcinolone acetonide by thin layer chromatography on silica gel using a developing

DAVID H.SIEH

630

solvent of benzene:methanol ( 8 5 : 1 5 ) and fluorescence at 366 nm. 6.54 High Performance Liquid Chromatography

Recently, a high-performance liquid chromatographic (HPLC) procedure involving the use of adsorption chromatography on Corasil I1 hasg4 been adopted by the United States Pharmacopeia for the determination of triamcinolone acetBgide with in 0.1 and 0.025% creams. A similar method a silica gel column and a solvent system of 95% ethano1:methylene chloride (5:95) has been used for the analysis of triamcinolone acetonide creams. Triamcinolone acggonide has also been determined in rice starch using adsorption HPLC. These methods, which do not involve the use of an internal standard, and numerous other methods for the detection and determination of triamcinolone acetonide with internal standards are listed below in Table 111.

Table 11.

Thin Layer Chromatography of Triamcinolone Acetonide

Solvent System

Adsorbent

Methylene Chloride: Silica gel methanol (15:6)

Detection

Rf

Radioactive scanner 0.91

Reference 79

A-Methylene Chloride: methanol ( 9 3 : 7 ) B-Cyclohexane: dioxane:water ( 5 0 :5 0 :10) Equal volumes of A and upper phase of B

0.36

Benzene:acetone (2:l)

0.22

Equal volumes of A and upper phase of B:methanol (9:2)

0.64

Methano1:ethyl (2:98)

Silica gel H

Radioactive scanner 0.45

30

Table 11.

Thin Layer Chromatography of Triamcinolone Acetonide (Continued)

Solvent System

Ad sorbe nt

Detection

Rf

To1uene:ethylacetate:85% formic acid (50:45: 5)

Kieselgel F-254

UV at 254 nm

0.38

To1uene:isopropanol: 37% ammonium hydroxide (70:29 :1)

0.59

To1uene:dioxane: methanol:37% ammonium hydroxide (20:50 :20: 10)

0.84

Methylene chloride: Silica gel GF 254 diethylether: methanol :water (77:15 :8 :l. 2) 1,l-dichloroethanol: acetone:acetic acid (160:40 :lo)

UV at 254 nm

0.45

0.42

Reference 78

79

Table 11.

Thin Layer Chromatography of Triamcinolone Acetonide (Continued)

Solvent System

Adsorbent

Detection

Rf

Reference

To1uene:chloroform (75:25) Stationary phaseF0rmamide:acetone (10:9 0 )

m

Ethylene dichloride: methano1:water ( 9 5 :5:0.2)

0.13

w

Ethylene chloride: methano1:water ( 9 5 :5 :0.2)

Silica gel

Butylacetate or Dichloroethane: methyl acetate : water (2:1:1), lower layer Stationary phase dioxane

Kieselgel 6 0 F 254

-

GF 254

5 0 % ethanolic

0.65

80

sulfuric acid, 12OOC f o r 5 minutes

relative to 168methylprednisolone acetate = 1.00

Spray with 20% anhydrous zinc chloride in methanol, llO°C for 20 minutes, UV at 254 and 3 6 6 nm

Identity Test

81

Table 11.

Thin Layer Chromatography of Triamcinolone Acetonide (Continued)

Solvent System

Adsorbent

Detection

Chloroform: methano1:glacial acetic acid (90:10:2)

Silica gel

W at 2 5 4 nm

0.47

82

To1uene:chloroform (75:25) Stationary phase-Formamide: acetone (10:90)

Kieselguhr G

Spray with 1 mg/ml sulfuric acid, violet color at 366 nm

0.39

67

Methylene chloride: Silica gel diethylether: methano1:water (77:15:8 :l.2) Chloroform: methano1:acetic acid (90:10:2)

Silica gel

Rf

0.13-0.15

G

Fluorescence at 2537AO

0.47

Reference

Table 111.

High Performance Liquid Chromatography of Triamcinolone Acetonide

Column

Mobile Phase

Flow Rate Retention Time Detection Reference (ml/min) (min) (nm)

16 reverse phase ODs, R P and phenyl columns

Acetonitrile: water (30:70)

2.0

Hypersil ODS 20 cm

Methanol: water (60:40)

1.0

10 p silica Gel 25 cm x 0.2 cm

95% ethanol: methylene chloride (5:95)

1.55

1.5

u Bondapak C18 Methano1:water 30 cm x 4.0 nun (70:30)

Methano1:water ( 5 0 :50)

Acetonitrile: water (60:40) Acetonitrile: water (40:60)

254

87

250

88

1.28

254

85

1.50

254

89

Variable

4.93 1.22 2.21 Re1ative to acetone 1.00

Table 111. Column

E m

High Performance Liquid Chromatography of Triamcinolone Acetonide (Continued) Mobile Phase

Flow Rate Retention Time Detection Reference (ml/min) (min) (nm)

Bondapak C18/ Methano1:water Corasil (60:40) 61 cm x 2.3 mm Methano1:water (40 :60) Acetonitrile: water (40:60) Acetonitrile: water (20:80)

1.5

Methano1:water Bondapak Phenyl/Corasil (60:40) Methano1:water (40:60) Acetonitrile: water (40:60) Acetonitrile: water (20:80)

1.0

Z ipax l m x 2 m m

0.33

watersaturated methy1ene chloride

1.22 6.41 1.11 8.12 Relative to acetone 1.00 1.28 5.40

1.17 4.56 Relative to acetone 1.00

7.2

254

86

T a b l e 111. Column

High Performance L i q u i d Chromatography o f Triamcinolone Acetonide (Continued) Mobile Phase

S p h e r i s o r b ODS Methano1:water 5-10 (56:44) 2 5 c m x 4.6 mm

8

I .

Petroleum Biosil A e t h e r :c h l o r o 20-44 5 0 cm x 2 . 1 mm form :methanol ( 6 0 :39:2) M et h y l e n e ch1oride:meth-

Flow Rate R e t e n t i o n T i m e D e t e c t i o n Reference (ml/min) (min) (nm) 1.3-1.5

1.0 or 250 kg/cm2

11.0

2 54

90,91

19.0

254

92

26.6

a n o l :water ( 9 7 :1:2)

M et h y l e n e

chloride: isopropanol (98:2)

13.3

Table 111.

High Performance Liquid Chromatography of Triamcinolone Acetonide (Continued)

Column

Mobile Phase

10% ODS p Bondapak

Acetonitrile: water (30:70)

Flow Rate Retention Time Detection Reference (ml/min) (min) (nm) 2.0

5% ODS Partisil

3-Rever sed Phase ODS columns

12.0

254

93

254

94

6.0

Acetonitrile: water ( 6 0 : 4 0 )

2.0

6.0

639

TRIAMCINOLONE ACETONIDE

Quantitative determination of triamcinolone acetonide in a 0.0152% aeresol spray $8 HPLC has Using been reported by Gibbs and Kirschbaum halcinonide as the internal standard, separation and quantitation was accomplished on a reverse phase Partisil ODS 25 cm x 4.6 mm column with a mobile phase of acetonitri1e:water (40:60), a flow rate of 1.0 to 1.5 ml/min and UV detection at 254 nm. 88 A procedure has been developed by Tenneson for the determination of triamcinolone acetonide in human serum by HPLC with a detection limit of 1 ng/ml. The triamcinolone acetonide is extracted from potassium chloride saturated serum samples into diethylether. After evaporation, the residue is taken up in the mobile phase (methanol:water, 60:40) and then separated (retention time 10 minutes) and quantified on a 5 micron Hypersil ODS HPLC column monitoring the absorbance of the eluate at 250 nm.

.

6.55 Gas Liquid Chromatography Three gas liquid chromatographic (GLC) systems for the separation and identification of triamcinolone acetonide are shown below in Table IV. In addition to those listed below, the gas chromatographic properties of triamcinolone acetonide that had been silanized at C-11 and disilanized at C-11 and C-21 have been studied by Triamcinolone acetonide was silanized Painter by reaction with trimethylsilylimidazo1e:bistrimethylsilyl acetamide:trimethylchlorosilane (3:3:2) and chromatographed on a 5 ft. x 0.020 in OV-1 stainless steel support coated open tubular column held at 220OC. The direct injector and flame ionization detector temperatures were 220 and 3OO0C, respectively. The helium carrier gas pressure was 5.0 psig. The disilanized product eluted at 2.5 minutes. The mass spectral characteristics of the silanized products were also determined (section 2.4).

.

Table IV.

GLC Systems for the Separation and Identification of Triamcinolone Acetonide Column T ("C)

Detection, carrier gas, flow rate

1% OV-17 on 100/120 silanized Supasorb 9 ft. x 0 . 2 5 in

260

EC, nitrogen, 100 cc/min

trimethylsilyl

31.5 min

96

3% OV-17 on Chromosorb WHP 1.8 m x 2.0 mm

260

FID,helium, 60 cc/min

trimethylsilyl

4.38 min

97

3% OV-3 on Chromosorb G

250

EC, nitrogen, 60 cc/min

trimethylsilyl

16 min

98

Column

Derivatization R,

Reference

TRIAMCINOLONEACETONIDE

641

6.56 Column Chromatography The versatile solvent system of n-hexane:ch1oroform:dioxane:water (90:10:40:5) was used for the partition chroygtographic isolation of triamcinolone acetonide The partition coefficients were determined on a diatomaceous earth column.

.

6.6

Fluorimetric Analysis

Kadin74 has described a fluorimetric de e mination of corticosteroids containing the A f 1 5 3-keto group. These A-ring dienone-containing steroids were reacted with zinc dust and 4 0 % sulfuric acid in n-butyl ether and the fluorescence emission and activation maxima determined at 390 and 340 nm, respectively. This simple method allowed the quantitation of triamcinolone acetonide in liquid formulations with a sensitivity of about 2 micrograms. Under the conditions employed only steroidal A-ring dienones fluoresce. Fluorescent silica gel chromatography and photodensitometry have been used to determine triamygnolone acetonide in pharmaceutical formulations

.

6.7

Titrimetric Analysis

Steroids with a 16a117a-diol group can be titrated with lead tetraacetate in acetic acid to a potentiometric endpoint. The micro version of this method using a 0.001 N lead tetraacetate solution enabled triamcinolone contam determined in triamcinolone acetonide%:3f to be 6.8

Differential Borohydride Analysis

Thel@,fferential borohydride assay developed by Gorag is an exc 1 ent method for the determination of A and A'1a-3-ketosteroids in pharmaceutical preparations. Addition of propylene glycol to the reaction mixturfO$o complex sodium to improve the metaborate allgwed Kir c baum reducti n of A - and A"'-3-ketosteroids. However l AP'4-3-ketosteroids i.e., triamcinolone were less than 10% reduced. By the use of lithium borohydride insbyad of sodium borohydride, Chafetz and co-workers were able to determine

-

DAVID H . SIEH

642

triamcinolone. This method should also allow the determination of triamcinolone acetonide. 6.9

Radioimmunoassay

A radioimmunoassay for triamcinolone acetonide &j plasma was described by Ponec and The antibody against triamcinolone co-workers acetonide was obtained by immunizing rabbits with triamcinolone acetonide-21-hemisuccinate coupled to bovine serum albumin. The minimum detectable amount was 200 pg.

.

7.

Determination in Body Fluids and Tissues

Triamcinolone acetonide has been determined in biological fluids and tissues by several different methods of analysis in combination with various extraction techniques. Table V summarizes the results that have been published since 1970.

Table V.

Determination of Triamcinolone Acetonide in Biological Fluids and Tissues

Chromatographic Method

Biological System

Extraction

Internal Standard

Re ference

GLC

Ethyl acetate Rat muscle followed by partitioning between hexane and 7 0 % aqueous methano1

3H -triamcinolone acetonide

96

GL.C

Ethyl acetate

Mouse and human dermal fibroblasts

Progesterone

97

Dichloromethane

Plasma and urine

H-triamcinolone acetonide

TLC with fluorescence

82,83

~~

HPLC

Diethylether

Human serum

88

644

8.

DAVID H. SIEH

Acknowledgements

The author would like to express his appreciation to Mr. Steve Highcock for conducting the literature search, to Dr. Mike Porubcan for determining and analyzing the nuclear magnetic resonance spectra, to Dr. John Dunham for his critical review of the manuscript and to Diane Walker for typing the manuscript. 9.

References

1. Florey, K., "Analytical Profiles of Drug Substances", Vol. 1, Academic Press, New York, N.Y., pp. 397-422 (1972). 2. Bellomonte, G., Ann. 1st. Super. Sanita, 9(part 2-31, 121-128 (1973). 3. Hahdlos, M., Arch. Pharm. Chemi., 82 (25-26) , 1392-1395 (1975). 4. Porubcan, M . , Squibb Institute for Medical Research, personal communication. 5. Blunt, J.W,, J.B. Stothers, Org. Magn. Res., 9(8), . _ 439 (1977). 6. Doddrell, D.M.,.D.T. Pegg, J. Amer. Chem. SOC., 102, 6388 (1980). 7. Lodge, B.A., P. Toft, J. Pharm. Pharmacol., 23 (31, 196-199 (1977). 8. Painter, J.L., Squibb Institute for Medical Research, personal communication. 9. Tomida, H., T. Yotsuyanagi, K. Ikeda, Chem. Pharm. Bull., 26(9), 2832-2837 (1978). 10. Grady, L.T., S.E. Hays, R.H. King, H.R. Klein, W.J. Mader, D.K. Wyatt, R.O. Zimmerer, Jr., J. Pharm. Sci., 62(3), 456-464 (1973). 11. Verma, S.C., J.O. Runikis, W.D. Stewart, Indian J. Hosp. Pharm., 10(5), 167-173 (1973) 12. Block, L.H., R.N. Patel, J. Pharm. Sci., 62 (4), 617-621 (1973). 13. Behl, C.R., L.H. Block, M.L. Borke, ibid., 65 (3), 429-430 (1976) 14. Yalkowsky, S.H. S.C. Valvani, ibid., 69(8), 912-922 (19801. 15. Flynn, GiL., ibid. , 60(3), 345-353 (1971). 16. Weber, D.J., T.R. Ennals, H. Mitchner, ibid., 61(5), 689-694 (1972). 17. Fairbrother, J.E., Methodol. Dev. Biochem., 5, 141-144 (1976).

.

-

645

TRIAMCINOLONEACETONIDE

18. Moellman, H . ,

B. Von Klot-Heydenfeldt, D.H. Miemeyer, H. A l f e s , I n t . 2 . K l i n . Pharmakol., Ther. Toxikol., 5(4), 434-443 (1972): C.A. 77:39117. 19. L i s t , . P . H . , J . M . Groenig, Pharm. I n d . ,

.

38 (12), 1159-1171 (1976) 20. Murata, R . , A. Tanuma, N . H i k i c h i , H . N i w a , Tohoku Yakka Daigaku Kenkyu Nempo, 25, 57-61 71978) ; CA 91:16297. 21. P a n i c h i , F . , S. A f r i c a n P a t e n t , 72-06559 (1973)i CA 80:37396. 22. P a n i c h i , F . , G e r . Offen., 2,243,480 (1974); CA 80:108759. 23. Toth, J . , A. Boor, M. K o v a t s i t s , 2 . Komesz, P. Major, T. Kovats, K . S . Gorgenyi, E. C z a j l i k , e t al, G e r . Offen., 2,246,203 (1973); CA 78:148133. 24. Reyba, S . A . , Spain P a t e n t , 414181 (1976); CA

85:177793. 25. C a s t e l l i , P . P . , A. A s c h e r i , G e r . Offen., 2, 448,548 (1975); CA 84:90423. 26. Rolland, G . I . , L. Mantica, R. Ciceri, G e r . Offen., 2,016,353 (1971); CA 75:47508. 27. Compania Espanola d e E s t e r o i d e s S.A., Spain P a t e n t , 483701 (1980); CA 93:9781. 28. Kupfer, D . , R. P a r t r i d g e , Arch. Biochem. Biophys., 140(1), 23-28 (1970). 29. Yamashita, O., Nippon Naibumpi Gakkai Z a s s h i , 47 (12), 1033-1045 (1972) 30. K r i p a l a n i , K . J . , A . I . Cohen, I. Weliky, E.C. S c h r e i b e r , J. Pharm. S c i . , 64(8), 1351-1359 (1975). 31. Gordon, S . , J. Morrison, S t e r o i d s , 32(1), 23-35 (1978). 32. Zimmerman, E . F . , D. Bowen, T e r a t o l o g y , 5(3), 335-343 (1972). 33. Zimmerman, E . F . , D. Bowen, i b i d . , 57-70 (1972). 34. K e i p e r t , J . A . , Med. J. Aust., 1(19), 1021-1025 (1971). 35. Kukita, A.. K . Yamada, T . Matsuzawa.

.

Y.

Takada, . Taehan Pibukwa Hakhoe Chapchi.

15(2), 115-122 (1977); CA 87:141187.

,

36. Hartmann, F . , P. Ude, Verh. Dtsch. G e s . I n n . Med., 80, 1548-1551 (1974); CA 83:6764. 37. Rasmussen, J . E . , Arch. Dermatol., 114, 1165-1167 (1978). 38. Woodford, R . , B.W. Barry, J. Pharm. S c i . , 66 (1), 99-103 (1977)

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DAVID H.SIEH

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39. Woodford, R., B.W. Barry, Curr. Ther. Res., Clin. Exp., 21(6), 877-886 (1977). 40. Vericat, C.F., C.R. Beaus, C.J. Coll, Con r

+

Nac. Biofarm. Farmacocinet., (Actas), 323-336 (1977); CA 89:135736. 41. Tronnier, H., Arch. Klin. Exp. Dermatol.,

237, 769-773 (1970). 42. Barry, B.W., A.R. Brace, J. Pharm. Pharmacol., 26(Supple), 67-68 (1974). 43. Schaefer, H., W. Schalla, Percutaneous Absorpt. Steroids (Int. Symp.) , 53-66 (1980) 44. Schaefer, H., G. Stuettgen, W. Schalla,

.

E. Bauer, J. Gazith, Ad;. Pharmacol. Ther., Proc. Int. Cong. Pharmacol., 7th, 9, 223 -235

71979). 45. Schaeffer, H., G. Stuettgen, A. Zesch, W. Schalla, J. Gazith, Curr. Probl. Dermatol., 7, 80-94 (19’78). 46. Schaeffer, H., G. Stuettgen, Mykosen, Suppl., 1, 164-170 (1978). 47. Schaeffer, H., A. Zesch, G. Stuettgen, Arch. Dermatol. Res., 258(3), 241-249 (1977). 48. Baker, J.R.J., R.A. Christian, P. Simpson, A.M. White, Br. J. Dermatol., 96(2), 171-178 (1977). 49. Lewis, J.D., B.D. Cameron, D.R. Hawkins,

L.F. Chasseaud. E.R. Frankline. Arzneim.-Forsch., 25(10), 1646-1650 (1975). 50. Ponec, M., M.K. Polano, Arch. Dermatol. Res.,

265, 101-104 (1979). 51. Rimbau, V., F. Lleonart, Ar2neim.-Forsch., 25 (7), 1042-1044 (1975) 52. Iyer, B.V., R.C. Vasavada, Int. J. Pharm., 3 (4-5), 247-260 (1979) 53. Iyer, B.V., Diss. Abstr. Int. B., 40(1), 173 (1979); CA 91:128981. 54. Polano, M.K., M. Ponec, Arch. Dermatol., 112 (5), 675-680 (1976) 55. Mezei, M., V. Gulasekharam, Life Sci., 26 (18), 1473-1477 (1980). 56. Gloor, M., J. Lindemann, Dermatol. Monatsschr., 116 (2), 102-106 (1980) 57. Lien, E.J., G.L. Tong, J. SOC. Cosmet. Chem., 24 (6), 371-384 (1973) 58. Hackney, J.F., S.R. Gross, L. Aronow, W.B. Pratt, Mol. Pharmacol., 6, 500-512 (1970).

.

. .

.

.

647

TRIAMCINOLONEACETONIDE

59. Biagi, G.L., A.M. Barbaro, 0. Gandolfi, M.C. Guerra, G. Cantelli-Forti, J. Med. Chem., 18 (91, 837-838 (1975). 60. Kakac, B., V. Rejholec, Cesk. Farm., 26(4) , 124-127 (1977). 61. Halot, D., M. Lanson, Ann. Pharm. Fr., 29 (11), 533-539 (1971) 62. Occhipinti, B., G. Rigamonti, M. Calati, Farmaco, Ed. Prat., 29(11), 611-620 (1974). 63. Tsuchikura, H., H. Shimano, T. Uete, Horumon To Rinsho., 18 (6), 501-504 (1970). 64. Koupparis, M.A., K.M. Walczak, H.W. Malmstadt, J. Pharm. Sci., 68(12), 1479-1482 (1979). 65. Yamashita, O., Y. Mineo, T. Nakagawa, K. Matsuoka, M. Nakamura, Y. Mishina, S. Marumoto, K. Okada, Horumon To Rinsho., 19 (7), 531-535 (1971); CA 77:1624 1. 66. Nakaji, Y., M. Ota, T. Hayakawa, J. Kawamura, Iyakuhin Kenkyu, 7 (1), 17-25 (1976); CA 88:141771. 67. Vanderhaeghe, H., J. Hoebus, J. Pharm. Belg., 31(1), 25-37 (1976). 68. Heintz, B., R. Kalusa, Dtsch. Apoth.-Ztg., 118(27), 1000-1006 (1978): CA 89:135905. 69. Szepesi; G., S. Gbrdg, Boll. Chim. Farm., 114 (2), 98-106 (1975) 7 0 . Chafetz, L., D.C. Tsilifonis, J. Pharm. Sci., 66 (8), 1145-1148 (1977) 71. Bundgaard, H., Arch. Pharm. Chemi. Sci. Ed., 6(3), 127-140 ('1978). 72. DeBoer, H.S., J. DanHartigh, H.H.J.L. Ploegmakers, W.J. Van Oort, Anal. Chim. Acta., 102, 141-155 (1978). 73. DeBoer, H.S., P.H. Lansaat, W.J. VanOort, ibid, 116 (1), 69-76 (1980) 74. Kadin, H., Microchem. J., 20(2), 236-241 (1975). 75. Massa, V., Labo-Pharma., 18(186), 80-81 (1970). 76. GbrOg, S. "The Analysis of Steroids", CRC Crit. Rev. Anal. Chem. , 9(4), 333-383 m 8 0 ) . 77. Czizier, E. S. Gorag, T. Szen, Microchim. Acta., 966 (1970). 78. Egli, R.A., S. Tanner, Fresenius 2. Anal. Chem., 295(5), 398-401 (19/9). 79. Cavina, G., N. Chemello, D. Loberto, I. Rocchi, E. Romaniello, L. Schweiger,

.

.

.

.

DAVID H.SIEH

648

G. Zanni, Ann. 1st. Super. Sanita, g(pt.41,

261-309 (1973). 80. Bailey, K., A.W. By, B.A. Lodge, J. Chromatogr., 166(1), 299-304 (197v. 81. Martin, J.L., R.E. Duncombe, W.H.C. Shaw, Analyst, 100 (1189), 243-248 (1975) 82. Kusama, M., N. Sakauchi, S. Kumaoka, Metab., Clin. Exp., 20(6), 590-596 (1971). 83. Kusama, M. N. Sakauchi, S. Kumaoka, Ni Naibumpi Gakkai Zasshi, 46(6), 654-6 (1970); CA 74 :109918. 84. United States Pharmacopeia, XX, 812 (1980). 85. Gaetani, E., C.F. Laureri, Farmaco, Ed. Prat., 29 (2), 110-118 (1974) 86. Higgins, J.W., J. Chromatogr., 115(1), 232-235 (1975). 87. Kirschbaum, J., R. Clay, R. Poet, "Symposium on the Analysis of Steroids", Eger, Hungary, May 1981, in press. 88. Tenneson, M.E. International Development Laboratory - E.R. Squibb and Sons, Inc., Personal communication. 89. Tymes, N.W., J. Chromatogr. Sci., 15(5), 151-155 (1977). 90. Gordon, G . , P.R. Wood, Proc. Anal. Div. Chem. SOC., 14(2), 30-32 (1977). 91. Gordon, G., P.R. Wood, Analyst, 101(1208), 876-882 (1976). 92. Cavina, G., G. Moretti, B. Gallinella, R. Alimenti, R. Barchiesi, Boll. Chim. Farm., 117 (9), 534-544 (1978). 93. Kirschbaum, J., J. Pharm. Sci., 69(4), 481-482 (1980). 94. KirschbaG, J,, R. Poet, K. Bush, G. Petrie, J. Chromatogr., 190(2), 481-485 (1980). 95. Gibbs, V., J. Krischbaum, Squibb Institute for Medical Research, personal communication. 96. Simpson, P.M., J. Chromatogr., 77(1), 161-174 (1973). 97. Au, D.S.-L., J.O. Runikis, F.S. Abbott, R.W. Burton, J. Pharm. Sci., 70(8), 917-923 (1981). 98. Goebbeler, K.H., J. Brienlich, Pharm.-Ztg., 28, 1037-1040 (1972) 99. Ponec, M., M. Frolich, A. DeLijster, A.J. Moolenar, Arch. Dermatol. Res., 259(1), 63-70 (1977). 100. GOrOg, S . , J. Pharm. Sci., 5 7 , 1137-1140 (1968).

.

P

.

.

TRIAMCINOLONE ACETONIDE

649

101. Chafetz, L., D.C. Tsilifonis, J.M. Riedl, ibid, 61, 148-150 (1972). 102. Kirschbaum, J., ibid, 67(2), 275-276 (1978). 103. Lerner, H., Squibb Institute f o r Medical Research, personal communication. 104. DeVincentis, J., Squibb Institute f o r Medical Research, personal communication.

TRIAMCINOLONE DIACEMTE David H.Sieh

652 652 652 655 655 656 656 656 656 657 657 677

1. Description 2. Physical Properties 2.2 Nuclear Magnetic Resonance

3. 6.

7. 8.

2.4 Mass Spectra 2.9 SolubilityData 2.10 Crystal Properties Synthesis Methods of Analysis 6.3 Colorimetric Analysis 6.5 Chromatographic Analysis Acknowledgements References

AnalyticalProfiles of Drug Substances Volume I 1

65 1

Copyright 0 1982 by The Americdn Phumaceutid Association

ISBN 012-2M8l1-9

DAVID H. SIEH

652

The following supplement contains updated information pertaining to the analytical chemistry of triamcinolone diacetate. A literature survey was conducted and is complete up to July 1981. The numbering system for topics discussed is the same as that in the original profile (Volume 1, pp. 4 2 2 - 4 4 2 ) .

1.

Description

Triamcinolone diacetate is a glucocorticoid used primarily in the treatment of adrenocortical and rheumatic disorders. 1.13 The CA Registry Number for triamcinolone diacetate is 67-78-7. 2.

Physical Properties 2.2

Nuclear Magnetic Resonance

2 The improved 'H-NMR and the 13C-NMR spectra of triamcinolone diacetate are shown in Figures 1 and 2 , respectively (please refer ty Figure 3 section 2 . 2 of the original profile for comparifyn and assignments of the 'H-NMR spectrum). The C-spectrum was determined on a JEOL FX60Q spectrometer using a 10 mm C/H dual probe. A compi9te assignment for all the carbon atoms in the C-NMR spectrum is listed in Table I. The assignments are based on relative chemfSallghifts C- F (dimethylsulfoxide-d = 39.5 PPM) and coupling constants akd are consistent wit3 literature values for closely related compounds The assignments were simplified by the utilization of polarization transfer to selectively enhance and phase alter individual carbon types

.

.

Figure 1. '€I-Nuclear magnetic resonance spectrum of triamcinolone 16,21diacetate (SQ 9465) in CDC13. Instrument: Varian XL-100A.

Figure 2. 13C-Nuclear magnetic resonance spectrum of triamcinolone 16,21diacetate (SQ 9465) in dimethylsulfoxide-d6. Instrument: JEOL FX60Q.

TRIAMCINOLONE DIACETATE

Table I.

655

I3C-NMR Spectral Assignmentsa of Triamcinolone 16,21-diacetate

Carbon Number

Chemigal Shift

Carbon Number

Chemica1 Shift

1 2 3 4 5 6 7 8 9 10 11 12

152.7 129.0 185.3 124.2 166.9 30.3 27.2 33.2(18.5)' lOl.O(l75.8) 48.0 (20.5) 70.4(36.1) 35.3

13 14 15 16 17 18 19 20 21 c=0 CH3

47.4 43.1 31.6 75.1 87.8 16.3 23.0 (4.9) 203.4 67.5 169.8 20.8 20.3

aAll chemical shifts are in PPM from tetramethylsilane with internal reierence dimethylsulwere run in foxide-d6 Z ~ ~ ~ . ~ ~ PAll P Mspectra . DMSO-d C- F coupling constants shown in parentfieses.

.

2.4

Mass Spectra

The low resolution mass spectra of 28 corticosteroid 21-esters and related compounds of pharmaceutical intefest have been determined by Toft and co-workers on an AEI MS-12 via direct probe. The mass spectra of triamcinolone and triamcinolofe diacetate are in excellent agreement with Florey

.

2.9

Solubility Data 2.91 Solubility

Solubilization of 19 steroid hormones, including triamcinolone, triamcinolone acetonide and triamcinolone diacetate by polyoxyethylene lauryl ethgr has been reported by Tomida and co-workers who also concluded that the solubilization of steroids by polyoxyethylene lauryl ether micelles was directly dependent upon their lipophilicity. Triamcinolone diacetate was found

DAVID H. SIEH

656

to have a solubility of 7.417x and 13.2 mg/g in 70% ethanol

.

M in water 6

2.92 Partition Coefficients The role of crystal structure (as reflected by the melting point and the entropy of fusion) and of the activity coefficient (as reflected by the octanol-water partition coefficient) in controlling the aqueous solubility of either liquid or crystalline organic nonelectrolytes, including triamcinolone, triamcinolone acetonide and triamcinolone diaGetate was discussed by Yalkowsky and Valvani The partition coefficients of triamcinolone diacetgte in two systems have been reported by He determined that the aqueous-micellar Tomida and octanol-water partition coefficients were 299 and 83.7, respectively. The value for the octanol-water partition coefficient is in excellent agreemeni-rath that published by other investigators

.

.

2.10

Crystal Properties

Murata and co-workers have reported the observation of crystal forms of triamcinolone diacetate and other glucocorticoids in sterile aqueous suspensions by scanning electron microscopy. 3.

Synthesis Higashikawa12 has patented a procedure for the preparation of triamcinolone diacetate by reacting the 21-hydroxy precursor with acetic anhydride in the presence of metal carbonate or hydroxide. A procedure for the synthesis of 16,17-dihydroxy steroid acetal and ketal derivatives anrj3acetylated compounds has been patented

.

6.

Methods of Analysis 6.3

Colorimetric Analysis

6.34 The reaction of a 1 mg/ml aqueous solution of sulfuric acid with triamcinolone diacetate produced a very faint color in daylight

657

TRIAMCINOLONE DIACETATE

anf4no color when viewed in the ultraviolet at 366 nm

.

6.37 Triamcinolone diacetate has been quantitated by its reaction with 2,3,5-triphenyltetrazoliumlt,jhloride followed by absorbance measurement

.

6.5

Chromatographic Analysis 6.52 Thin Layer Chromatographic Analysis

Four systems used for the separation and identification of triamcinolone diacetate by thin layer chromatography are shown in Table 11. 6.54 High Performance Liquid Chromatography A compilation of the liquid chromatographic separation systems for triamcinolone diacetate published after 1970 is shown in Table 111. K' is defined as the capacity ratio.

7.

Acknowledgements

The author would like to express his appreciation to Mr. Steve Highcock for performing the literature search, to Dr. Mike Porubcan for determining and analyzing the nuclear magnetic resonance spectra, and to Dr. John Dunham for his critical review of the manuscript. 8. 1.

2. 3. 4. 5. 6.

References Florey, K., "Analytical Profiles of Drug Substances", Vol. 1, Academic Press, New York, N.Y., pp. 422-442 (1972). Porubcan, M., Squibb Institute f o r Medical Research, personal communication. Blunt, J.W., J.B. Stothers, Org. Magn. R e s . , 9(8), 439-444 (1977). Doddrell, D.M., D.T. Pegg, J. Amer. Chem. SOC., 102, 6388-6391 (1980) Toft, P., B . A . Lodge, M.B. Simard, Can. J. Pharm. Sci., 7(2), 53-61 (1972). Tomida, H., T. Yotsuyanagi, K. Ikeda, Chem. Pharm. Bull., 26 (9), 2832-2837 (1978).

.

TABLE 11.

Thin Layer Chromatography of Triamcinolone Diacetate

Solvent System

Adsorbent

Detection

R,

Reference

&

Methylene chloride: Silica gel dioxane: water (10:5:5)- GF 254 lower layer

Spray with 50% 0.88 sulfuric acid, heat at 12OOC for 5 min. 1.00 Re1ative to 168methyl prednisolone acetate-1.00

Ethylene chloride: methanol: water (95:5:0.2)

Chloroform Stationary phase Formamide: acetone (10:90) Methylene chloride: diethy1ether:methanol: water (77:15:8 :l.2 )

16

Kieselguhr

G

Silica Gel

G

Spray with 1 mg/ml sulfuric acid, uv at 366 nm

0.18

0.27

14

Table 111. Diacetate.

m

$

High Performance Liquid Chromatographic Systems for Triamcinolone Flow Retention Rate Time (ml/min) (min)

Detection

Column

Mobile Phase

Zipax lm x 2mm

Water saturated methylene chloride

0.33

4.8

254

17

IJ Bondaapak c18 30cm x 3.9 mm

Acetonitrile: (30:70)

1.0

K'=8.48

240

18

l~ Bondapak c18 30 cm x 4.0mm

Methanol :water (70 :30) 1.5 Methano1:water (50:50) Acetonitrile:water(60:40) Acetonitrile:water(40:60)

1.30 3.38 1.25 2.65 Relative to acetone 1.00

254

19

Bondapak C / Corasif 61 cm x 2.3 mm

Methanol :water ( 60:40) Methano1:water (40:60) Acetonitrile:water(40:60) Acetonitrile:water(20:80)

1.33 4.88 1.11 9.06 Acetone = 1.00

water

(nm)

Reference

Table 111. Diacetate.

High Performance Liquid Chromatographic Systems for Triamcinolone Flow Rate (ml/min)

Column

Mobile Phase

BondapakPhenyl/ Corasil 61 cm x 2.3 mm

Methano1:water (60:40) 1.0 Methanol :water ( 4 0 : 60) Acetonitrile:water(40:60) Acetonitrile:water(20:80)

Retention Time (min) 1.21 5.60 1.21 6.31 Acetone = 1.00

Detection (nm)

Reference

66 1

TRIAMCINOLONEDIACETATE

7. 8.

Grady, L.T., S.E. Hays, R.H. King, H.R. Klein, W . J . Mader, D.K. Wyatt, R.O. Zimmerer, Jr., J. Pharm. S c i . , 6 2 ( 3 j , 456-464 ( 1 9 7 3 ) . Verma, S.C., J . O . Runikis, W.D. Stewart, Indian J. Hosp. Pharm., 1 0 ( 5 ) , 167-173 (1973).

Yalkowsky, S . H . , S.C. Valvani, J . Pharm. Sci., 6 9 ( 8 ) , 912-922 ( 1 9 8 0 ) . 1 0 . Flynn, G.L., ibid., 6 0 ( 3 ) , 345-353 ( 1 9 7 1 ) . 11. Murata, R . , A. Tanuma, N. Hikichi, H. Niwa, Tohoku Yakka Daigaku Kenkyu Nempo, 2 5 , 57-61 ( 1 9 7 8 ) ; C.A. 91:16297. 1 2 . Higashikawa, T., Patent 5 5 4 0 6 3 0 6 5 , 5 5 4 0 6 3 0 6 6 48160B. ( 1 9 7 9 1 , Derwent 41 3 . Compania Espanola de Esteroides S.A., Spain Patent, 4 8 3 7 0 1 ( 1 9 8 0 ) ; C.A. 93:9781. 1 4 . Vanderhaeghe, H., J. Hoebus, J. Pharm. Belg., 31 (1), 25-37 ( 1 9 7 6 ) 15. Heintz, B., R. Kalusa, Dtsch. Apoth.-Ztg., 1 1 8 ( 2 7 ) , 1000-1006 ( 1 9 7 8 ) ; C.A. 8 9 : 1 3 5 9 0 5 . 1 6 . Bailey, K., A.W. By, B.A. Lodge, J. Chromatogr., 1 6 6 (1), 299-304 ( 1 9 7 8 ) 1 7 . Higgins, J.W., ibid., 1 1 5 ( 1 ) , 232-235 ( 1 9 7 5 ) . 1 8 . Van Dame, H.C., J. Assoc Off. Anal. Chem., 9.

.

.

6 3 ( 6 ) , 1184-1188

19.

(1980).

Tymes, N.W., J. Chromatogr. Sci., 1 5 ( 5 ) , 151-155

(1977).

CUMULATIVE INDEX Bold numerals refer to volume numbers

Acetaminophen, 3, 1 Acetohexamide, 1, 1; 2,573 Allopurinol, 7, 1 Alpha-tocopheryl acetate, 3, 111 Aminophylline, L1, 1 Aminosalicylic acid, 10, 1 Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 Amphotericin B, 6, 1; 7, 502 Ampicillin, 2, I; 4, 518 Ascorbic acid, ll, 45 Aspirin, 8, 1 Azathioprine, 10, 29 Bacitracin, 9, 1 Bendroflumethiazide, 5, I; 6, 597 Benzyl benzoate, 10, 55 Betamethasone dipropionate, 6, 43 Bretylium tosylate, 9, 71 Bromocriptine methanesulfonate, 8, 47 Calcitriol, 8, 83 Captopril, ll, 79 Carbamazepine, 9, 87 Cefaclor, 9, 107 Cefamandole nafate, 9, 125; 10, 729 Cefazolin, 4, 1 Cefotaxime, ll, 139 Cefoxitin, sodium, 11, 169 Cephalexin, 4,21 Cephalothin sodium, 1, 319 Cephradine, 5,21 Chloral hydrate, 2, 85 Chloramphenicol, 4,47, 518 Chlordiazepoxide, 1, 15 Chlordiazepoxide hydrochloride, 1, 39; 4, 518 Chloroquine phosphate, 5, 61 Chlorpheniramine maleate, 7.43

Chloroprothixene, 2, 63 Chlortetracycline hydrochloride, 8, 101 Clidinium bromide, 2, 145 Clindamycin hydrochloride, 10, 75 Clofibrate, 11, 197 Clonazepam, 6, 61 Clorazepate dipotassium, 4, 91 Clotrimazole, 11, 225 Cloxacillin sodium, 4, 113 Codeine phosphate, 10, 93 Colchicine, 10, 139 Cyanocobalamin, 10, 183 Cyclizine, 6, 83; 7, 502 Cycloserine, 1, 53 Cyclothiazide, 1, 66 Cypropheptadine, 9, 155 Dapsone, 5.87 Dexamethasone, 2, 163; 4,519 Diatrizoic acid, 4, 137; 5, 556 Diazepam, 1, 79; 4, 518 Dibenzepin hydrochloride, 9, 181 Digitoxin, 3, 149 Digoxin, 9,207 Dihydroergotoxine methanesulfonate, 7, 81 Dioctyl sodium sulfosuccinate, 2, 199 Diperodon, 6, 99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disulfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Dopamine hydrochloride, 11, 257 Doxorubicine, 9, 245 Droperidol, 7, 171 Echothiophate iodide, 3,233 Emetine hydrochloride, 10, 289 Epinephrine, 7, 193 663

664 Ergonovine maleate, 11, 273 Ergotamine tartrate, 6, 113 Erythromycin, 8, 159 Erythromycin estolate, 1, 101; 2, 573 Estradiol valerate, 4, 192 Ethambutol hydrochloride, 7, 231 Ethynodiol diacetate, 3, 253 Fenoprofen calcium, 6, 161 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 281 Flufenamic acid, 11, 313 Fluorouracil, 2, 221 Fluoxymesterone, 7, 251 Fluphenazine decanoate, 9,275; 10, 730 Fluphenazine enanthate, 2, 245; 4, 524 Fluphenazine hydrochloride, 2. 263; 4. 519 Flurazepam hydrochloride, 3, 307 Gentamicin sulfate, 9, 295; 10; 731 Glibenclamide, 10, 337 Gluthethimide, 5, 139 Gramicidin, 8, 179 Griseofulvin, 8, 219, 9, 583 Halcinonide, 8, 251 Haloperidol, 9, 341 Halothane, 1, 119; 2, 573 Heroin, 10, 357 Hexestrol, 11, 347 Hexetidine, 7, 277 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide, 10,405 Hydroflumethiazide, 7, 297 Hydroxyprogesterone caproate, 4, 209 Hydroxyzine dihydrochloride, 7, 319 Iodipamide, 3, 333 Isocarboxazid, 2,295 Isoniazide, 6, 183 Isopropamide, 2, 315 Isosorbide dinitrate, 4, 225; 5, 556 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Ketoprofen, 10,443 Khellin, 9, 371 Leucovorin calcium, 8, 315 Levarterenol bitartrate, 1, 49; 2, 573; 11, 555 Levallorphan tartrate, 2, 339 Levodopa, 5. 189 Levothyroxine sodium, 5, 225 Lorazepam, 9.397 Meperidine hydrochloride, 1, 175 Meprobamate, 1, 209; 4, 520; 11, 587 6-Mercaptopurine, 7, 343 Mestranol, 11, 375

CUMULATIVE INDEX Methadone hydrochloride, 3, 365; 4, 520; 9,601 Methaqualone, 4,245, 520 Methimazole, 8, 351 Methotrexate, 5, 283 Methoxsalen, 9, 427 Methyclothiazide, 5, 307 Methylphenidate hydrochloride, 10,473 Methyprylon, 2, 363 Metronidazole, 5, 327 Minocycline, 6, 323 Nabilone, 10,499 Nadolol, 9,455; 10, 732 Nalidixic acid, 8, 371 Natamycin, 10, 513 Neomycin, 8, 399 Nitrazepam, 9,487 Nitrofurantoin, 5, 345 Nitroglycerin, 9, 519 Norethindrone, 4, 268 Norgestrel, 4, 294 Nortriptyline hydrochloride, 1,233; 2, 573 Noscapine, 11,407 Nystatin, 6, 341 Oxazepam, 3,441 Oxytocin, 10, 563 Penicillamine, 10, 601 Penicillin-G benzothine, 11, 463 Penicillin-V, 1,249 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4, 319; 5,429 Phenobarbital, 7, 359 Phenoxymethyl penicillin potassium, 1, 249 Phenylbutazone, ll,483 Phenylephrine hydrochloride, 3, 483 Piperazine estrone sulfate, 5, 375 Primidone, 2, 409 Probenecid, 10, 639 Procainamide hydrochloride, 4, 333 Procarbazine hydrochloride, 5, 403 Promethazine hydrochloride, 5,429 Proparacaine hydrochloride, 6,423 Propiomazine hydrochloride, 2,439 Propoxyphene hydrochloride, 1,301; 4,520; 6, 598 Propylthiouracil, 6, 457 Pseudoephedrine hydrochloride, 8,489 Reserpine, 4, 384; 5, 557 Rifampin, 5, 467 Salbutamol, 10, 665 Secobarbital sodium, 1, 343

CUMULATIVE INDEX Spironolactone. 4,431 Sodium nitroprusside, 6, 487 Succinylcholine chloride, 10,691 Sulfadiazine, 11, 523 Sulphamerazine, 6, 515 Sulfamethazine, 7, 401 Sulfamethoxazole, 2, 467; 4, 521 Sulfasalazine, 5, 515 Sulfisoxazole, 2, 487 Testolactone, 5, 533 Testosterone enanthate, 4, 452 Theophylline, 4, 466 Thiostrepton, 7,423 Tolbutamide, 3, 513; 5, 557 Triamcinolone, 1, 367; 2, 571; 4, 521, 524; 11,593

Triamcinolone acetonide, 1, 397, 416; 2, 571; 4, 521,7, 501; 11,615

665

Triamcinolone diacetate, 1, 423; 11, 651 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2, 507 Trifluoperazine hydrochloride. 9, 543 Triflupromazine hydrochloride, 2, 523; 4, 521; 5,557

Trimethaphan camsylate, 3. 545 Trimethobenzamide hydrochloride, 2, 551 Trimethoprirn, 7,445 Trioxsalen, 10,705 Triprolidine hydrochloride, 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7,477 Tybamate, 4, 494 Valproate sodium and valproic acid, 8, 529 Vinblastine sulfate, 1,443 Vincristine sulfate, 1,463